1

RESEARCH ARTICLE 1 2

The reverse-transcriptase/RNA-maturase protein MatR is required for the 3 splicing of various group II introns in Brassicaceae mitochondria 4

5 Laure D. Sultan1,+, Daria Mileshina2,+, Felix Grewe3,4 , Katarzyna Rolle5, Sivan Abudraham1, Paweł 6 Głodowicz5, Adnan Khan Niazi2, Ido Keren1, Sofia Shevtsov1, Liron Klipcan6,7, Jan Barciszewski5, 7 Jeffrey P. Mower3, André Dietrich2, Oren Ostersetzer-Biran1,* 8

9 1 Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences, 10 The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel.11 2 Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général 12 Zimmer, 67084 Strasbourg, France. 13 3 Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA 14 4 Current Address: Integrative Research Center, The Field Museum, 1400 S Lake Shore Drive, Chicago, 15 IL 60605, USA 16 5 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, 17 Poland 18 6 Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel 19 7 Current Address: Institute of Plant Sciences, the Gilat Research Center, Agricultural Research 20 Organization (ARO), Rural Delivery Negev, Israel. 21 + These authors contributed equally to this work.22 * Corresponding Author: oren.ostersetzer@mail.huji.ac.il.23

24 Short title: MatR role in mitochondria group II intron splicing 25

One-sentence summary: MatR, a highly conserved, essential mitochondrial protein, functions in the processing and maturation of various pre-RNAs in plant mitochondria, as revealed by in vivo analyses.

The author responsible for the distribution of materials integral to the findings of this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Oren Ostersetzer-Biran (oren.ostersetzer@mail.huji.ac.il.).

26 ABSTRACT 27 Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal 28 introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are 29 particularly prevalent within plants organellar genomes. Proteins encoded within the introns 30 themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns 31 in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, 32 only a single maturase has been retained in the mitochondrial DNA: the matR gene found within 33 NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing 34 events, which restore conserved amino acids, indicates that matR encodes a functional protein. 35 However, the biological role of MatR remains unclear. Here, we performed an in vivo 36 investigation of the roles of MatR in Brassicaceae. Directed-knockdown of matR expression via 37 synthetically designed ribozymes altered the processing of various introns, including nad1 i4. 38 Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other 39 intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual 40 evolutionary transition from the intron-specific maturases in bacteria into their versatile 41

Plant Cell Advance Publication. Published on October 19, 2016, doi:10.1105/tpc.16.00398

©2016 American Society of Plant Biologists. All Rights Reserved

2

spliceosomal descendants in the nucleus. The similarity between maturases and the core 42 spliceosomal Prp8 protein further support this intriguing theory. 43 44

INTRODUCTION 45

Mitochondria play central roles in cellular energy production and metabolism. As progenies from 46

a free-living prokaryotic symbiont, mitochondria contain their own genomes (mtDNAs), 47

ribosomes and proteins. While the mtDNAs in animal cells are typically small (16–19 kbp), 48

encoding 37 or fewer tightly packed genes, the mtDNAs in plants are notably larger (100–11,300 49

kbp) and variable in their structure (Kubo and Newton, 2008; Maréchal and Brisson, 2010; 50

Arrieta-Montiel and Mackenzie, 2011; Knoop, 2012; Sloan et al., 2012; Small, 2013; Gualberto 51

et al., 2014). In angiosperms, the mtDNAs contain about 60 identifiable genes encoding tRNAs, 52

rRNAs, ribosomal proteins and various subunits of the energy transduction pathway, but they 53

also harbor numerous open reading frames (ORFs), many of which are not conserved between 54

different species and whose functions are currently unknown (reviewed in e.g. (Mower et al., 55

2012). Our work focuses on the analysis of a maturase-related (MatR) ORF, which is encoded 56

within the fourth intron in NADH dehydrogenase 1 (nad1) (Wahleithner et al., 1990). 57

The expression of the mitochondrial genomes in plants is complex, particularly at the 58

post-transcriptional level (see e.g. (Liere et al., 2011; Small et al., 2013; Hammani and Giege, 59

2014). To become functional, the primary organellar transcripts undergo extensive processing 60

events, including RNA editing (commonly C-to-U conversions) and the splicing of numerous 61

introns that reside within the coding regions of many essential genes (Bonen, 2008; Brown et al., 62

2014; Schmitz-Linneweber et al., 2015). These essential processing steps are regulated by 63

various protein cofactors, which may link the respiratory-mediated functions with environmental 64

or developmental signals (see e.g. (Li-Pook-Than et al., 2004; Dalby and Bonen, 2013). Several 65

of the factors required for mitochondrial introns splicing have been identified, but there are 66

certainly many more to be discovered (Brown et al., 2014; Schmitz-Linneweber et al., 2015). 67

With the exception of the horizontally acquired group I intron in the cox1 (cytochrome c 68

oxidase subunit 1) gene of some angiosperms (Vaughn et al., 1995; Sanchez-Puerta et al., 2008), 69

all other mitochondrial introns in angiosperms are classified as group II-type (Bonen, 2008). 70

Canonical introns belonging to this class are self-catalytic RNAs (i.e. ribozymes) and mobile 71

genetic elements, which are defined by a highly conserved secondary structure of six stem-loop 72

3

domains (DI - DVI) radiating from a central RNA core (Michel et al., 1989; Ferat and Michel, 73

1993). Based on their structural features and splicing chemistry (i.e. two consecutive 74

transesterification steps, with a bulged A from the intron acting as the initiating nucleophile), 75

group II introns are proposed to be the progenitors of spliceosomal RNAs (reviewed in e.g. 76

(Cech, 1986). Although some model group II introns are able to catalyze their own excision in 77

vitro, independently of proteins (Michel et al., 1989; Ferat and Michel, 1993; Lambowitz and 78

Belfort, 1993; Saldanha et al., 1993; Michel and Ferat, 1995), the conditions for self-splicing are 79

generally non-physiological (i.e. high temperatures and salt conditions), and for their efficient 80

splicing in vivo, the group II-encoding pre-RNAs depend upon interactions with various 81

proteinaceous cofactors (Lambowitz and Belfort, 2015). In bacteria and yeast mitochondria, 82

proteins that function in the splicing of group II introns typically involve maturases (MATs), 83

which are encoded within the fourth stem-loop domain (DIV) of the introns themselves. The 84

MATs were shown to bind with high affinity and specificity to their own cognate intron-RNAs 85

and are postulated to facilitate intron splicing by assisting the folding of these highly structured 86

RNAs into their catalytically active forms under physiological conditions (Cousineau et al., 87

1998; Wank et al., 1999; Singh et al., 2002; Noah and Lambowitz, 2003; Cui et al., 2004; 88

Blocker et al., 2005; Huang et al., 2005; Ostersetzer et al., 2005). 89

Group II MATs contain several conserved motifs that are required for both splicing and 90

intron mobility (Mohr et al., 1993; Michel and Ferat, 1995; Wank et al., 1999; Matsuura et al., 91

2001; Aizawa et al., 2003; Cui et al., 2004; Lambowitz and Zimmerly, 2011; Lambowitz and 92

Belfort, 2015; Zimmerly and Semper, 2015). These include a region with sequence similarity to 93

retroviral-type reverse transcriptases (i.e. the RT domain), with conserved sequence blocks that 94

are present in the fingers and palm regions of retroviral RTs, and a conserved sequence motif 95

similar to the thumb domain of retroviral RTs (also denoted as domain X), which is associated 96

with RNA binding and splicing (Mohr et al., 1993). In addition to the RT domain, these proteins 97

may harbor C-terminal DNA binding (D) and endonuclease (En) domains that are found in some, 98

but not all, of the MATs. 99

Phylogenetic studies show that the organellar introns in plants have all evolved from 100

MAT-containing introns (Bonen and Vogel, 2001; Toor et al., 2001; Zimmerly et al., 2001; 101

Ahlert et al., 2006; Lambowitz and Belfort, 2015; Zimmerly and Semper, 2015). Yet, only a 102

single mat-related gene has been retained in each of the organelles in angiosperms, namely, matK 103

4

found in the trnK intron in the plastids (Mohr et al., 1993; Hausner et al., 2006) and a 104

mitochondrial maturase-related ORF (matR), encoded within the fourth intron of the nad1 gene 105

(i.e. nad1 i4), encoding subunit 1 of respiratory complex I (Wahleithner et al., 1990). Both MatK 106

and MatR are well conserved between different monocot and dicot species, and are thus expected 107

to retain similar functions in all angiosperms. 108

Several lines of evidence suggest that MatK is involved in the splicing of plastidial group 109

II introns in plants (Zoschke et al., 2009; Schmitz-Linneweber et al., 2015). Plants with defective 110

plastidial ribosomes are unable to splice the subclass group IIA introns in the chloroplast 111

(Jenkins et al., 1997). Also, several parasitic plants that lack matK have also lost the plastid-112

encoded group IIA introns from their plastid DNAs (Funk et al., 2007; Wicke et al., 2013), while 113

plants that contain matK as a stand-alone ORF have retained some subgroup IIA introns in their 114

reduced plastid genomes (Wolfe et al., 1992; Ems et al., 1995). Recently, using transplastomic 115

lines in tobacco (Nicotiana tabacum), (Zoschke et al., 2010) and his colleagues showed that 116

MatK is associated in vivo with many of the subgroup IIA introns in the chloroplasts (Zoschke et 117

al., 2010). These data strongly support the notion that MatK plays a prominent role in plastid 118

RNA metabolism. 119

Likewise, the roles of MatR are also expected to be essential for mitochondrial biogenesis 120

in plants. MatR is closely related to maturases encoded by group II introns in bacteria 121

(Wahleithner et al., 1990) and has been retained as a conserved ORF in the mtDNAs in nearly all 122

angiosperms (Adams et al., 2002). Different RNA analyses show that the matR transcripts 123

undergo several RNA-editing events, which restore conserved amino acids (Thomson et al., 124

1994; Begu et al., 1998). Yet, as a transformation system has not been established for 125

mitochondria in plants, no loss- or gain-of-function studies are currently available to test this 126

theory. In this report, we use Arabidopsis thaliana (Col-0) and cauliflower (Brassica oleracea 127

var. botrytis), two key members of the Brassicaceae family, to study the functions of MatR in 128

plants. While Arabidopsis serves as a prime model system for plant molecular genetics, 129

cauliflower is employed for biochemical analysis of plant mitochondrial RNA metabolism 130

(Neuwirt et al., 2005). We present several lines of evidence indicating that the matR locus 131

encodes a mitochondrial protein, which is associated with various pre-RNAs in Brassicales. We 132

further implemented an innovative system for genetic manipulation of mitochondrial gene 133

expression in Arabidopsis using synthetic ribozymes (Val et al., 2011) and show that a reduction 134

5

in MatR expression affects the processing of a number of group II introns in Arabidopsis 135

mitochondria. 136

137

RESULTS 138

matR gene structure in Brassicales 139

In Brassicaceae, as in many plant species, the nad1 gene is fragmented into five exons in mtDNA 140

(see e.g. (Grewe et al., 2014). These are typically expressed as three individual transcription 141

units, which are fragmented in two of the introns (i.e. nad1 i1 and nad1 i3). For its maturation, 142

nad1 undergoes the splicing of two trans-spliced pre-RNAs (i.e. nad1 i1 and nad1 i3) and the 143

excision of two introns (nad1 i2 and nad1 i4) that are found in a classical cis configuration. The 144

fourth intron in nad1 (i.e. nad1 i4) encodes a well conserved maturase-related reading frame, 145

denoted as MatR (Wahleithner et al., 1990; Chapdelaine and Bonen, 1991; Thomson et al., 146

1994). Figure 1A shows a schematic secondary structure model of nad1 i4 and its cognate 147

maturase-related (matR) gene, in Arabidopsis. Notably, sequencing data indicate that while nad1 148

i4 is found in a standard ‘cis’ configuration in Brassicales (Grewe et al., 2014), in some plant 149

species, such as petunia, tobacco, wheat and rice, nad1 i4 is fragmented in DIV either upstream 150

or downstream of the matR reading frame (Figure 1B; and (Malek et al., 1997; Bonen, 2008). 151

The nature of the complex biochemical interactions between group II introns and their 152

protein partners is under investigation. The best characterized maturase to-date is LtrA maturase 153

from the bacterium Lactococcus lactis, which binds with high affinity and specificity to its host 154

intron, the Ll.LtrB pre-RNA. The LtrB group II intron is able to self-splice in the presence of 155

high Mg2+ concentrations in vitro, but its splicing at physiological conditions in vivo requires the 156

LtrA protein (Saldanha et al., 1999). The association of LtrA with the LtrB intron is facilitated 157

primarily through its association with regions found within DIV of the intron (Wank et al., 158

1999). The binding of LtrA with regions in LtrB intron DIV may enable its association with core 159

intron elements, promoting short- and long-range tertiary RNA base interactions that are 160

necessary for RNA folding and catalysis (Matsuura et al., 2001; Noah and Lambowitz, 2003). 161

Based on the PHYRE (Kelley and Sternberg, 2009) and ROBETTA (Kim et al., 2004) 162

servers, the MatR protein in Brassicales shares similarity with the reverse transcriptase (RT) 163

domains of various proteins, including telomerases (c3du6A; confidence 99.6), HIV-1 reverse-164

transcriptases (c1rthA; confidence 97.3) and DNA/RNA polymerases (d1ztwa1; confidence 165

6

96.2). The RT domains of model group II-encoded MATs contain several subdomains, which are 166

required for RNA recognition and intron splicing (see e.g. (Lambowitz and Belfort, 2015; 167

Zimmerly and Semper, 2015; Piccirilli and Staley, 2016; Qu et al., 2016; Zhao and Pyle, 2016). 168

These include the finger and palm subdomains of a polymerase and the less conserved domain X 169

that is analogous to a polymerase thumb domain. In addition, model maturases also contain in 170

the RT region an N-terminal subdomain (NTD), which in the case of the bacterial LtrA protein is 171

required for the binding of the maturase with regions in DIV of its host ltrB intron RNA that 172

include the LtrA-ORF’s Shine-Dalgarno site (Qu et al., 2016). This suggests that canonical 173

MATs may autoregulate their own expression in vivo through binding to their own ribosome 174

binding sites (see e.g. (Matsuura et al., 2001; Singh et al., 2002). 175

Analysis of conserved domains, implemented in the NCBI server (Marchler-Bauer et al., 176

2015), showed that the MatR-ORF in Arabidopsis harbors only some parts of the consensus RT 177

region, i.e. sequence blocks RT 3 to 7 of the finger-palm and an intact thumb (X) motif, but does 178

not contain an N-terminus NTD subdomain (Figure 2). Like the nucleus-encoded type I 179

maturases (Mohr and Lambowitz, 2003; Keren et al., 2009), MatR also lacks the C-terminal 180

DNA binding and endonuclease (D/En) domains. It is therefore anticipated that the intron 181

mobility functions associated with some canonical group II intron-encoded MATs has been lost, 182

while splicing activity is retained for mitochondrial MatR in plants (see e.g. (Brown et al., 2014; 183

Schmitz-Linneweber et al., 2015). Interestingly, MatR harbors two large insertions in the finger-184

palm region, between the conserved sequence blocks RT-4 and RT-5 (amino acids 153-317) and 185

between the RT-7 and the thumb motif (amino acids 385-533), which may correspond to 186

additional RT subdomains, which are unique to the MatR protein (Figure 2, labeled by dotted 187

boxes, and Supplemental Figure 1). 188

189

RNA-seq data indicate that the matR gene is transcribed as part of a polycistronic unit in 190

Brassicales mtDNAs 191

To analyze the expression of MatR in Brassicaceae, we used different RNA methodologies, i.e. 192

RNA-seq (high-throughput RNA sequencing), RACE (rapid amplification of cDNA ends) and 193

RT-PCR analyses, with purified mitochondrial RNAs (mt-RNAs) preparations. For this purpose, 194

we used mitochondria isolated from cauliflower (Brassica oleracea var. botrytis) inflorescences, 195

which allows the purification of large quantities of highly enriched organellar preparations from 196

7

a plant that is also closely related to Arabidopsis (Neuwirt et al., 2005; Keren et al., 2009). 197

Previously, we established the genome sequence of Brassica oleracea mitochondria (Grewe et 198

al., 2014). Comparisons of the mtDNA sequences of Arabidopsis and cauliflower show that nad1 199

i4, and its cognate matR reading frame, are nearly identical between the two plant species 200

(Supplemental Figure 1). 201

RNA-seq of total cauliflower mt-RNA suggested that matR is found on a large 202

polycistronic transcript unit containing the nad1 i4 sequence together with nad1 exons 4 and 5, a 203

(pseudo) gene-fragment of nad5 (i.e. ψnad5-2), and possibly also several ORFs of unknown 204

functions (i.e. orf159, orf161 and orf287) (Supplemental Figure 2). RNA-seq also indicated the 205

presence of U bases at many positions in the RNA sequence where the DNA sequence has C 206

bases (Supplemental Figure 2, labeled in green). These likely correspond to RNA editing sites, 207

which seem to be particularly prevalent in the coding regions of nad1 exons 4 and 5, but also 208

within the sequence corresponding to the ψnad5-2 gene. The putative RNA editing sites in the 209

nad1 pre-RNA are also shown in Supplemental Figure 3. 210

We further performed RACE to characterize the putative position of the 5’ and 3’ ends of 211

the nad1 i4 pre-RNA transcripts. Using circularized mt-RNAs as templates for the RACE 212

analyses (Eyal et al., 1999; Forner et al., 2007), the sequencing data of the RACE products 213

identified only mitochondrial transcripts that harbored the matR gene together with the upstream 214

regions of nad1 i4, the fourth exon in nad1 (i.e. nad1 ex4), and a stretch of 120 additional 215

nucleotides (i.e. the longest RACE sequence identified) upstream to nad1 ex4 belonging to the 216

3’-end of the trans-spliced nad1 i3 (see Supplemental Figure 3). No transcripts containing only 217

the matR-ORF could be identified by the RACE analyses. The 3’ end of the transcript included 218

about 240 nucleotides downstream of nad1 ex5. 219

Comparisons of the deduced MatR protein sequences from various plants indicate a high 220

flexibility of the N-terminal domains (Supplemental Figure 1). The upstream region of the 221

canonical start site of the MatR sequences contains putative codons that are also recognized as a 222

part of the conserved RT-2 sequence block of group II intron-encoded MATs (Figure 2 and 223

Supplemental Figure 1). Interestingly, different editing sites identified by the RNA-seq and 224

RACE data also included a C-to-U substitution 28 nucleotides upstream of the first common 225

AUG, found inside this putative RT-2 motif of MatR-ORF (Supplemental Figure 3). It will 226

therefore be interesting to investigate whether the translation of MatR may initiate upstream to 227

8

the first common AUG site. Although translation from non-AUG codons has not yet been proven 228

for mitochondria in plants, there is some indirect evidence indicating that GTG and AUA can 229

initiate translation for several mitochondrial genes in different plant species (see e.g. (Bock et al., 230

1994; Siculella et al., 1996; Zhu et al., 2014). 231

The maturation of pre-RNAs in plant organelles involves a complex series of post-232

transcriptional maturation events, which include 5' and 3' end processing and numerous 233

nucleation events that lead to the release of the mRNAs from their corresponding polycistronic 234

transcripts (see e.g. (Germain et al., 2013; Small et al., 2013; Brown et al., 2014; Hammani and 235

Giege, 2014). While the putative 5′ and 3’ terminal regions of nad1 i4 are likely generated post-236

transcriptionally, no RNAs corresponding to the MatR coding region alone, nor to any 237

alternative matR transcripts other than the nad1 ex4-i4-ex5 pre-RNA, were supported by the 238

RNA-seq and RACE data. These observations may imply that, similar to the bacterial maturases 239

(reviewed by (Lambowitz and Zimmerly, 2011; Zimmerly and Semper, 2015), MatR translation 240

is facilitated by mitoribosomes that are directly associated with the pre-RNA of nad1 i4. Yet, the 241

data are not sufficient to assess whether translation is initiated by non-AUG start codons. Studies 242

are underway to test this intriguing possibility. 243

244

matR encodes a ~70 kDa protein that is associated with mitochondrial membranes 245

We anticipate that the matR locus encodes a functional protein, but this notion was not yet tested 246

experimentally. MatR was not found among the different proteins identified in global mass-247

spectrometry (LC/MS-MS) analyses of plant organellar preparations (Jacoby et al., 2010; Taylor 248

et al., 2011; Lee et al., 2012; Huang et al., 2013; Nelson et al., 2013; Nelson et al., 2014; 249

Shingaki-Wells et al., 2014). However, this may be expected due to its predicted low expression 250

level in the mitochondria, as was also evident in the cases of MatK (Zoschke et al., 2010) and the 251

nucleus-encoded (nMAT) homologs (Keren et al., 2009; Keren et al., 2012; Cohen et al., 2014), 252

as well as various other RNA-processing enzymes in the mitochondria in angiosperms (Law et 253

al., 2012). 254

As no antibodies to MatR protein were available prior to this study, a polyclonal antibody 255

was raised against a synthetic peptide corresponding to a unique region in the RT domain, which 256

is conserved among different Brassicales (i.e. NH3-RRIDDQENPGEEASFNA) (Supplemental 257

Figure 1). The affinity and specificity of the antibody was confirmed in protein gel-blots using 258

9

total protein and enriched organelle preparations (i.e. mitochondria, chloroplasts and 259

peroxisomes) obtained from 3-week-old Arabidopsis thaliana var. Columbia (Figure 3). The 260

immunoblots with the anti-MatR antibodies showed a single band corresponding to a protein of 261

about 70 kDa, which is also the expected size for a native MatR protein (calculated mass 73.65 262

kDa, assuming that MatR translation initiates at the first common AUG site; see Supplemental 263

Figure 1); this was observed only in the mitochondria subfraction (Figure 3A). The use of 264

antibodies to the mitochondrial proteins serine-hydroxymethyltransferase (SHMT) and 265

cytochrome oxidase subunit 2 (COX2), the plastid-encoded 33 kDa subunit of the oxygen 266

evolving complex of PSII (PsbO) and the peroxisomal marker catalase (CAT) protein confirmed 267

that the organelle preparations were highly enriched (Figure 3A). 268

We further investigated whether MatR is found in the soluble (matrix) or membranous 269

fractions in the mitochondria. For this purpose, intact mitochondria (Figure 3B) were disrupted 270

by several freeze and thaw cycles followed by sonication and then separated by centrifugation 271

into the pellet, which contains the membranes (M) and the supernatant (S) containing the matrix 272

components. The fractionation procedure was validated by protein gel blot analyses with 273

antibodies against SHMT (as an indicator of matrix proteins), while COX2 was used as a marker 274

of organellar (inner) membrane proteins (Figure 3B). Unexpectedly, the immunoblot assays 275

show that the MatR protein is enriched in the mitochondrial membranous fraction (Figure 3B), 276

even though the deduced protein sequence of MatR lacks any obvious membrane targeting 277

motifs. 278

To investigate the topology of MatR in the mitochondria, the membrane fraction was pre-279

washed with 10 mM Tris-HCl pH 8.0 (to remove contaminating proteins) and then treated with 1 280

M NaCl (removes loosely attached membrane proteins), 2 M NaBr (a chaotropic agent used for 281

the removal of some peripherally-bound proteins), or 0.05% (v/v) Triton X-100 + 1 M NaCl (this 282

low concentration of the detergent will not completely solubilize the membranes but can wash 283

peripheral proteins attached by surface hydrophobic interactions) (Ostersetzer et al., 2007). The 284

data indicate that MatR is likely to be peripherally associated with the membranes, as neither the 285

10 mM Tris-HCl nor the 1 M NaCl treatments released the protein from the membranes, whereas 286

treating the organellar membranes with 2 M NaBr or 0.05% Triton X-100 + 1 M NaCl removed 287

the MatR signal from the membranes (Figure 3C). Similarly to MatR, the peripheral AtpB 288

subunit of the mitochondrial ATP-synthase coupling factor-1 (CF1) was removed by either 2 M 289

10

NaBr or 0.05% Triton X-100 + 1 M NaCl, but not by the 10 mM Tris-HCl or 1 M NaCl washes. 290

In contrast to MatR and AtpB, the 2 M NaBr wash did not remove the signals of the Nad9 291

subunit of the soluble arm of complex I or the COX2 protein of complex IV, which is integrally 292

associated with the mitochondrial inner-membranes. While the Nad9 subunit was largely 293

removed from the membranes by 0.05% Triton X-100 + 1 M NaCl, the integral COX2 protein 294

was only partially affected by the detergent treatment (Figure 3C). Taken together, these results 295

indicate that the MatR protein associates peripherally with membranes. As MatR is synthesized 296

inside the organelles, we speculate that the protein is associated with the organellar inner 297

membranes. 298

299

MatR expression profiles in Arabidopsis thaliana 300

The relative accumulation of transcripts corresponding to matR and nad1 in Arabidopsis was 301

analyzed by RT-qPCR in different plant tissues and during different development stages (Figure 302

4). The data indicate that the steady-state levels of transcripts corresponding to matR/nad1-i4 and 303

to the mature nad1 exons 4-5 were all below detectable levels in dry (desiccated) seeds. Notably, 304

accumulation of transcripts corresponding to matR/nad1 i4 pre-mRNAs was evident as early as 2 305

hours after initial seed imbibition (Figure 4). The steady-state levels of matR pre-RNA 306

transcripts gradually increased during the imbibition stage, reaching the highest level between 12 307

to 48 hours after initial water uptake by the dry seed. Based on the RNA-seq and RACE data, it 308

is anticipated that matR is co-transcribed with nad1 i4. The subtle differences in the 309

accumulation of transcripts corresponding to matR and nad1 i4 pre-RNA may be due to the 310

differences caused by the RT-qPCR analyses, i.e. the use of different sets of oligonucleotides or 311

the differences in the sizes of the PCR products (153 nts and 64 nts, respectively) (see Figure 4). 312

Following seed initiation, where the radicle protrudes through the seed coat (i.e. about 48 hours), 313

the levels of matR transcripts steadily decreased throughout the plant’s development. In contrast 314

to the matR expression profiles, accumulation of mature nad1 RNA transcripts corresponding to 315

ex4-5 was apparent only after ~24 hours of seed imbibition (Figure 4). Following this step, the 316

steady-state levels of nad1 ex4-5 mRNA gradually increased, peaking around 10 days after seed 317

initiation (Figure 4). 318

To analyze the accumulation of MatR and various other mitochondrial proteins during 319

early seed germination, total protein was extracted from dry and imbibed Arabidopsis seed 320

11

following 1 and 2 days of imbibition, and the accumulation of mitochondrial proteins was 321

examined by SDS-PAGE and protein gel blotting analyses (Figure 5). The immunoblots 322

indicated that the ~70 kDa band, corresponding to the MatR protein, was hardly detectable in the 323

dry seeds, whereas it was clearly visible in the imbibed seeds after 24 hours and further increased 324

in its levels after 48 hours of imbibition. However, the MatR protein signal was noticeably 325

reduced (about 2.5x lower) in mature Arabidopsis seedlings (i.e. at 1.0x dilution) compared with 326

the levels of MatR in germinating seeds two days following the initial imbibition stage (Figure 327

5A). The levels of the outer mitochondrial membrane protein porin-1 (VDAC1) were also higher 328

(1.3~1.8x) in seeds than in mature (i.e. 3-week-old) seedlings. In Arabidopsis, VDAC1 has a 329

ubiquitous expression pattern but shows particularly high expression levels in reproductive 330

organs and during germination and early seedling establishment (Pan et al., 2014). 331

The levels of several other mitochondrial proteins, including Nad9 and the nucleus-332

encoded CA2 (carbonic anhydrase like subunit 2) of complex I (CI), the Rieske Iron-Sulfur 333

protein (RISP) of CIII, the COX2 subunit of CIV, and the AtpB subunit of the ATP-synthase 334

enzyme (CV) also increased during the imbibition stage (Figure 5A). Yet, unlike the patterns of 335

MatR and VDAC1 accumulation, the steady-state levels of these proteins were not significantly 336

altered during the first 24 hours of seed imbibition and increased only 48 hours following initial 337

seed imbibition. Unlike MatR and VDAC1, the highest steady-state levels of the respiratory 338

subunits Nad9, CA2, RISP, COX2 and AtpB, were observed in mature plants rather than seeds 339

(Figure 5A). Blue-native (BN)-PAGE gel electrophoresis, followed by protein gel blot analyses, 340

corroborated the immunoblot results. The mitochondrial holo-complexes corresponding to CI, 341

CIII, CIV and CV had the lowest abundances in dry seeds, but then gradually increased in their 342

levels during the 48 hours of imbibition, showing the highest abundances in mature (i.e. 3-week-343

old) seedlings (Figure 5B). The numbers below the blots in Figure 5 show the relative 344

accumulation (quantified using ImageJ software (Jensen, 2013) of different organellar proteins 345

during the imbibition stage and in mature, 3-week-old Arabidopsis seedlings. 346

347

MatR is associated with various pre-RNAs in Brassicales mitochondria 348

It was previously noted that group II intron-containing pre-RNAs are associated with their 349

corresponding splicing cofactors in bacteria and chloroplasts in plants (Stern et al., 2010; Barkan, 350

2011; Lambowitz and Zimmerly, 2011; Germain et al., 2013). Likewise, the mitochondrial 351

12

maturase-related nMAT2 protein is bound to several group II RNAs, which were also identified 352

as the native intron targets of the protein in vivo (Keren et al., 2009). These data suggest that 353

splicing cofactors of group II introns are associated specifically with those introns whose 354

splicing they facilitate in bacteria and in plant organelles. Accordingly, co-immunoprecipitation 355

(co-IP) analyses with antibodies raised against different organellar splicing factors indicated that 356

the plant factors typically co-precipitated with their ‘genetically-defined’ intron targets (see e.g. 357

(Ostheimer et al., 2003; Schmitz-Linneweber et al., 2005; Schmitz-Linneweber et al., 2006; 358

Asakura and Barkan, 2007; Watkins et al., 2007; Keren et al., 2009; Zoschke et al., 2010). Here, 359

we further applied this approach to define and catalog the native RNA targets of MatR protein. 360

For this purpose, anti-MatR antibodies were used in pull-down experiments to pellet the 361

associated MatR-RNP complexes from solubilized (1% NP-40, v/v) mitochondria preparations 362

obtained from cauliflower inflorescences. cDNAs prepared from the RNA recovered by the co-363

IPs were applied to custom arrays and analyzed by hybridization to specific intron probes (see 364

Methods; and Supplemental Figure 4). Commercial antibodies against the mitochondrial SHMT 365

protein (Supplemental Table 1) were used as a control for the RNA co-366

immunoprecipitation/microarray (RIP)-chip analysis. RNAs identified by the MatR-RIP-chip 367

assay included nad1 i4, the host intron of matR, as well as several other organellar transcripts, 368

including nad1 i1, nad1 i3, nad4 i1, nad5 i4, nad7 i2, rpl2 i1 and rps3 i1 (Figure 6 and 369

Supplemental Figure 4). In addition to these RNAs, weaker signals were seen in the cases of 370

nad2 i2, i3 and i4 and nad4 i2 (Supplemental Figure 4). The signals of abundant organellar 371

transcripts, such as tRNAs, rRNAs and many of the organellar mRNAs, were all below 372

detectable levels, strongly supporting the specificity of the RIP-chip analyses. 373

The association of MatR with pre-RNAs corresponding to nad1, nad4, nad5, rpl2 and 374

rps3 was also supported by sequencing analyses of cDNA libraries prepared from the pelleted 375

RNAs obtained from the co-IPs with MatR antibodies (Supplemental File 1). Therefore, we 376

assume that the nad1, nad5, nad7, rpl2 and rps3 pre-RNAs represent the native intron targets of 377

the MatR protein in Brassicales mitochondria. Unlike MatR, no RNAs were identified in the 378

SHMT-related RIP-chip analysis (Supplemental Figure 4). 379

In addition to identifying and characterizing mt-RNAs that are associated with the MatR 380

protein, we also characterized different proteins that are found together with MatR in 381

mitochondrial ribonucleoprotein particle(s) in vivo (i.e. proteins that have been co-precipitated 382

13

with MatR in the RIP-chip assays). LC/MS-MS analyses of MatR indicated the presence of 383

several mitochondrial proteins that co-precipitated with MatR (Supplemental Table 2). Several 384

proteins, such as heat shock proteins, prohibitins, and ATP-synthase subunits, are abundant in 385

the mitochondria and may therefore be non-specifically precipitated by the MatR antibodies 386

(some were also identified by the co-IPs with SHMT antibodies). Yet, in addition to MatR, other 387

proteins identified by the co-IPs may correspond to splicing cofactors that are associated together 388

with MatR in splicesomal-like RNP particles in the mitochondria (see e.g. (Schmitz-Linneweber 389

et al., 2015). Such proteins may include the splicing cofactor DEAD-box ATP-dependent RNA 390

helicase 53 (PMH2) (Köhler et al., 2010) and a few other RNA-binding cofactors belonging to 391

the large family of the pentatricopeptide repeat-containing (PPR) proteins in plants (Small and 392

Peeters, 2000) (Supplemental Table 2). Importantly, these data indicate that polypeptides 393

corresponding to the MatR protein are indeed present in angiosperm mitochondria. 394

395

Down-regulation of MatR expression by a pioneering ribozyme-based strategy in 396

Arabidopsis mitochondria 397

The RIP-chip data indicate that MatR is associated with many mitochondrial pre-RNAs (Figure 6 398

and Supplemental Figure 4). However, these data are not sufficient to indicate a direct role for 399

MatR in the processing of group II introns in angiosperms mitochondria. As no methods are 400

currently available to modify mtDNA in plants, we sought to utilize a modified hammerhead 401

ribozyme system, which was recently shown to cleave and thereby strongly down-regulate the 402

accumulation of mitochondrial atp9 transcripts in vivo (Val et al., 2011). To investigate the roles 403

of MatR in the processing of group II intron-containing transcripts in plant mitochondria, 404

synthetically engineered trans-cleaving hammerhead ribozymes (matRz1 and matRz2), designed 405

to cleave inside the matR reading frame, were driven into the organelles as "passenger" 406

molecules conjugated to a tRNAVal-like shuttle RNA ‘vector’ (Figure 7; and (Val et al., 2011). 407

Because the functions of MatR may be essential to the plant, the chimeric constructs, comprising 408

the matR-ribozyme components fused to the tRNA-like shuttle vector, were cloned under an 409

estradiol-inducible promoter (Zuo et al., 2000). To assess possible side effects caused by the 410

expression of mitochondria-localized ribozymes, we further designed a ribozyme against the 411

tobacco (Nicotiana tabacum) mitochondrial sdh3 gene (Figure 7), which is not present in the 412

mitochondria of Brassicaceae, including Arabidopsis. The transgenic lines matRz1 and matRz2 413

14

represent individual transformants expressing different synthetic ribozymes that were designed to 414

affect the expression of the Arabidopsis thaliana matR gene in two separate gene-loci (see Figure 415

7). 416

Upon induction of the matRz ribozymes, the levels of matR transcripts were reduced by 417

50–60% in the matRz1 and the matRz2 lines, as determined by RT-qPCR analyses (Figure 8 and 418

Supplemental Figure 5). The steady-state levels of the matR transcripts remained low for at least 419

5 days following estrogen induction of the transgenes. Accordingly, the steady-state levels of 420

MatR protein also decreased and was reduced by ~50% in matRz1 and by ~20% in matRz2 after 421

5 days following the induction of the matRz ribozymes (Figure 8 and Supplemental Figure 5). No 422

significant changes in MatR accumulation and expression were seen in the control sdhRz lines 423

(Figure 8). Ribozyme-mediated knockdown of matR had no significant effect on the steady-state 424

levels of various intronless mitochondrial RNAs (see Supplemental Figure 5). Under normal 425

growth conditions (see Methods), expression of the ribozymes generated no obvious phenotypic 426

differences between the transgenic lines and wild-type plants (i.e. within the time-frame 427

considered) (Supplemental Figure 6). This may be expected due to only partial reductions in 428

MatR expression in the two matRz lines (Figure 8 and Supplemental Figure 5), while the 429

complete loss of matR gene function is expected to be lethal to the plant. 430

The accumulation of each of the 23 group II introns in Arabidopsis mitochondria (Unseld 431

et al., 1997), including nad1 i1-i4, nad2 i1-i4, nad4 i1-i3, nad5 i1-i4, nad7 i1-i4, cox2 i1, ccmFc 432

i1, rpl2 i1, rps3 i1 and their flanking exons, was analyzed in the matRz and sdhRz lines at the 433

time of induction (Figure 9A) and 5-days (Figure 9B) following the addition of estrogen to the 434

growth medium. The expression profiling of the Arabidopsis mt-RNAs by RT-qPCR was 435

performed generally as described previously (Colas des Francs-Small et al., 2012; Keren et al., 436

2012; Zmudjak et al., 2013; Cohen et al., 2014). Oligonucleotides designed for the different exon 437

and intron sequences are indicated in Supplemental Table 3A. Introns that were notably affected 438

by the reduction in MatR expression (i.e. at day 5) included the sole introns within rpl2, rps3, 439

cox2 and ccmFc (all in a ‘cis’ configuration) as well as nad1 i3 (trans-spliced), nad1 i4 (cis-440

spliced), nad5 i4 (cis-spliced), and nad7 i2 (cis-spliced) (Figure 9). To a lesser extent, reduced 441

splicing efficiencies (i.e. higher pre-RNA to mRNA ratios) were also seen in the cases of nad2 i4 442

(cis-spliced), nad4 i3 (cis-spliced) and nad5 i3 (trans-spliced). In each case, the accumulation of 443

unspliced pre-mRNAs was correlated with decreased transcript levels in the corresponding 444

15

mRNAs, in both matRz1 and matRz2 lines, compared with those of the control sdhRz plants 445

(Figure 9 and Supplemental Figure 7). Accumulation of pre-mRNAs in the matR-reduced lines 446

was also apparent for nad2 i4, nad4 i3 and nad5 i3 (Figure 9B). However, as the corresponding 447

mRNA levels (i.e. spliced exons) were not significantly affected in the matRz lines 448

(Supplemental Figure 7), we could not draw any firm conclusions regarding the putative roles of 449

MatR in the splicing of these introns. The levels of mt-RNAs corresponding to nad2 exons 2-3, 450

exons 3-4 (and to some extent nad2 exons 4-5, as well), and nad5 exons 1-2 were somewhat 451

higher in the matRz lines, while no significant differences in the transcript levels of nad1 i1 and 452

i2, nad2 i1, nad4 i1, nad7 i1, i3 and i4, or their corresponding mRNAs (i.e. spliced exons) were 453

seen between the sdhRz and matRz lines (Figure 9 and Supplemental Figure 7). Thus, we 454

conclude that the splicing of this entire subset of mitochondrial RNAs does not require MatR. 455

456

DISCUSSION 457

Angiosperm genomes encode several MAT-related proteins, which function in the splicing 458

of different subsets of organellar group II introns 459

Sequences similar to retroviral-type reverse transcriptases (RT) are widely distributed among 460

different organisms. In bacteria, such genes include various ORFs encoded within many of the 461

group II introns termed as maturases (MATs) (Reviewed in e.g. (Lambowitz and Zimmerly, 462

2011; Lambowitz and Belfort, 2015; Zimmerly and Semper, 2015). These were shown to act 463

specifically in the splicing and intron mobility (i.e. retrohoming) activities of their own cognate 464

intron RNAs. While the chemistry of the splicing reaction is mediated by the introns themselves, 465

mounting evidence indicates that the MATs are required to facilitate or to stabilize the folding of 466

the highly structured group II RNAs into their catalytically active forms. 467

Plant genomes encode several proteins that are closely related to bacterial group II 468

reverse transcriptase/maturase proteins (Figure 3) (Mohr and Lambowitz, 2003; Knoop, 2012; 469

Brown et al., 2014; Schmitz-Linneweber et al., 2015). In angiosperms, these include MatK 470

encoded by the group II intron of trnK (i.e. tRNALys UUU) in the plastids (Mohr et al., 1993), 471

four nucleus-encoded maturases (i.e. nMAT1 to 4) (Mohr and Lambowitz, 2003) that were all 472

shown to reside in the mitochondria in Arabidopsis (Keren et al., 2009), and the mitochondrial 473

MatR ORF found inside nad1 i4 (Wahleithner et al., 1990) that is in the focus of this work. 474

16

The six maturases in angiosperms are all expected to function in the splicing of different 475

subsets of group II introns in plant organelles (Schmitz-Linneweber et al., 2015). Genetic and 476

biochemical studies indicate a role for MatK in the processing of about half of the plastidial 477

introns, all belonging to sub-group IIA (Zoschke et al., 2010). Likewise, the functions of the 478

nucleus-encoded nMAT homologs, including nMAT1, nMAT2 and nMAT4, are required for the 479

splicing of various group II introns in the mitochondria, many of which reside within RNAs 480

encoding complex I subunits (Nakagawa and Sakurai, 2006; Keren et al., 2009; Keren et al., 481

2012; Cohen et al., 2014). It is therefore anticipated that other plant maturases, including the 482

nucleus-encoded nMAT3 and the mitochondria-encoded MatR proteins, will also function in the 483

splicing of mitochondrial pre-RNAs in plants. Further investigations of the roles of nMAT3 are 484

underway in our laboratories. The matR sequence has remained intact in the mtDNAs of nearly 485

all angiosperms and gymnosperms (Guo and Mower, 2013). Furthermore, analyses of the 486

transcription profiles of many plant species indicate that matR transcripts undergo various RNA-487

editing events that restore conserved amino acids (see also Supplemental Figure 3). Here, we 488

provide multiple lines of evidence indicating that the matR locus encodes a protein that functions 489

in the processing of many mitochondrial group II intron-containing RNAs in Brassicales. 490

491

The 3D homology model of MatR reveals similarities with canonical bacterial maturases 492

A major advance in the understanding of the functionality of maturases (MATs) in group II 493

intron splicing has been recently accomplished by structural analyses of bacterial MATs bound 494

to their cognate group II intron RNAs (Piccirilli and Staley, 2016; Qu et al., 2016; Zhao and 495

Pyle, 2016). These including the crystal structures of the RT domains of MATs from the bacteria 496

Roseburia intestinalis and Eubacterium rectale (Zhao and Pyle, 2016), and a cryo-EM analysis 497

of the ribonucleoprotein complex of the Lactobacillus lactis intron-encoded LtrA maturase 498

bound to its host ltrB intron RNA (Qu et al., 2016). Analyses of the structures of the spliced ltrB 499

intron (at 4.5 Å resolution) and of the ltrB intron in its ribonucleoprotein complex with LtrA (at 500

3.8 Å resolution) are further revealing functional coordination between the intron RNA with its 501

cognate MAT-protein. Remarkably, these structures reveal close relationships between the RT 502

catalytic domain and telomerases, whereas the ‘active splicing centers’ resemble that of the Prp8 503

protein (Dlakic and Mushegian, 2011; Galej et al., 2013; Yan et al., 2015), which also resides at 504

the core of the spliceosome. 505

17

We tested whether MatR in plants may adopt a structural fold similar to that of bacterial 506

maturases (e.g. LtrA protein). Using the ‘RaptorX’ web-server (Kallberg et al., 2012), we 507

constructed a homology 3D model of the Arabidopsis MatR protein (Supplemental Figure 8) in 508

an attempt to map functional domains in MatR and to address its putative roles in mitochondria 509

group II intron RNA recognition and splicing. The predicted structure of MatR includes most of 510

the regions of the fingers-palm motif and the thumb (X) domain (Supplemental Figure 8). As 511

MatR shows only limited homology in the C-terminal region to model maturases, this region 512

could not be fully modeled. We were also unable to provide a structural prediction of the two 513

insertions in MatR found inside the fingers-palm (Figure 2 and Supplemental Figure 8, labeled 514

with dotted lines) due to the lack of homology of these regions with known protein sequences. 515

Nonetheless, the superimposed 3D model of MatR protein fits quite well with the established 516

structures of the bacterial LtrA maturase (Supplemental Figure 8). 517

We further speculate that predicted basic surfaces in MatR (i.e. positively charged 518

regions; see Supplemental Figure 8, regions labeled in blue) may correspond to RNA-binding 519

sites required for the association of the protein with its target pre-RNA molecules, in a similar 520

manner to the bacterial maturases (Qu et al., 2016; Zhao and Pyle, 2016). However, additional 521

structural studies and biochemical assays would be required to confirm the hypothetical 3D 522

model of MatR protein structure and to analyze its binding characteristics. 523

524

The N-terminal region of MatR: Potential use of alternative translation initiation sites for 525

MatR proteins in angiosperms mitochondria 526

Comparison of the deduced amino-acid sequences of MatR-ORFs from different angiosperms 527

indicates a promiscuous mode of translational initiation. Some, including MATs in dicots, most 528

monocots, and a few rosids, begin with an ‘AGA’ triplet (for arginine) followed by 18 putative 529

‘codons’ upstream of the first common initiation (AUG) site (i.e. 530

RKKEGLKFRLTVVLPIEKIM), while other MatR-ORFs in eudicots (e.g. Nicotiana tabacum) 531

begin with the ‘GGG’ triplet (for glycine) (i.e. RKK*GSKFRPLTVVLPIEKIM), and a stop 532

codon is found at the 4th codon position in the matR’s coding region (Supplemental Figure 1). 533

Interestingly, the codons upstream of the canonical start site are recognized as part of the 534

conserved RT-2 sequence block of group II maturases (Figure 2 and Supplemental Figure 1). 535

The translation of some nuclear genes in eukaryotes can initiate at codons that differ from the 536

18

consensus site in one base (e.g. CUG, ACG, AUU and GUG) (Kozak, 1989; Gordon et al., 1992; 537

Hann, 1994; Kozak, 1997). Prokaryotes and mitochondria in animals use alternative initiation 538

codons more frequently (commonly U/GUG in bacteria and AUA/U in animal mitochondria) 539

(Lobanov et al., 2010). Although the translation from non-AUG codons has not yet been proven 540

for mitochondria in plants, there is some indirect evidence strongly indicating that GUG and 541

AUA can initiate translation for several mitochondrial genes in different plant species (Bock et 542

al., 1994; Siculella et al., 1996; Zhu et al., 2014). 543

544

The expression of MatR is developmentally regulated 545

Gene expression mechanisms that occur in plant mitochondria include RNA editing, splicing of 546

many group II introns, and numerous nucleation events that lead to the release of mRNAs from 547

their organellar-encoded polycistronic transcripts (see e.g. (Germain et al., 2013; Small et al., 548

2013; Brown et al., 2014; Hammani and Giege, 2014). The MatR ORF in angiosperm 549

mitochondria is found within the fourth intron in nad1. An uncertainty regarding MatR 550

expression in angiosperms involves the transcription of the gene by an alternative promoter site 551

found inside nad1 i4, upstream from the matR locus. In wheat, this may lead to the expression of 552

a truncated MatR product, which lacks most of the N-terminal RT domain (Farre and Araya, 553

1999). The expression of MatR in Arabidopsis and cauliflower was studied here by different 554

RNA and protein methodologies. RNA-seq and RACE analyses (Supplemental Figure 2 and 3) 555

indicated that MatR is co-transcribed together with its host nad1 i4, but failed to reveal the 556

presence of any transcripts containing only the MatR ORF, arguing against the presence of any 557

internal transcriptional start sites in cauliflower. As MatR is most likely required for the 558

maturation of its own transcript, we speculate that MatR is translated directly from the unspliced 559

precursor nad1 ex4-i4-ex5 transcript, in a similar manner to the canonical maturases encoded by 560

group II introns in bacterial genomes (Lambowitz and Zimmerly, 2011; Zimmerly and Semper, 561

2015). 562

The immunoblot assays with anti-MatR antibodies identified a single protein of ~70 kDa 563

in the mitochondria, which corresponds to an intact MatR protein (Figure 3A). No additional 564

protein bands corresponding to polypeptides of either lower or higher molecular weight were 565

seen in the protein gel blot analyses. In the mitochondria, MatR was found to be associated 566

mainly with the membranes (Figure 3B). Analysis of MatR topology by different salt and 567

19

detergent treatments indicated that the protein is most likely peripherally bound to the 568

membranes (Figure 3C). The association of MatR with the membranes is surprising, especially 569

as its plastidial homolog, MatK, is found in the stromal chloroplast fraction (Zoschke et al., 570

2010). Thus, it remains possible that under the low salt conditions used for mitochondrial sub-571

fractionation (see the Methods section), MatR interacts non-specifically with the negatively 572

charged organellar membranes. Alternatively, the binding of MatR with the membranes might be 573

facilitated by its association with membrane-bound ribonucleoprotein particle(s) (see e.g. 574

(Uyttewaal et al., 2008). 575

The accumulation of MatR and several other mitochondrial proteins was studied during 576

early developmental stages in Arabidopsis (Figure 4 and 5). The data indicate that the expression 577

and accumulation of MatR, at both the RNA and protein levels, is upregulated during early seed 578

imbibition, but declines progressively after the initiation stage as plant development proceeds. 579

These data suggest that the MatR protein is unstable and undergoes degradation via the 580

proteolytic machinery in the mitochondria. The levels of VDAC1 were also higher in 581

germinating seeds compared mature (i.e. 3-week-old) seedlings. In Arabidopsis, VDAC1 is 582

required for maintaining the steady state of mitochondrial transmembrane potential (ΔΨ) and 583

hence for optimal respiratory activities, and its functions were found to be critical during the 584

reproductive stage and during early germination (Pan et al., 2014). In contrast to MatR and 585

Porin, the steady-levels of various other mitochondrial proteins, including Nad9, CA2, RISP, 586

COX2 and AtpB, all increased in during plant development, with the highest accumulation levels 587

seen in mature plants. In accordance with these data, faster turnover rates were linked to proteins 588

involved in DNA/RNA metabolism and the stress response in the mitochondria, whereas 589

subunits of the respiratory machinery are generally more stable in Arabidopsis cells (Nelson et 590

al., 2013; Nelson et al., 2014). 591

592

Unlike canonical maturases, MatR is involved in the splicing of many group II-containing 593

pre-RNAs in Brassicales mitochondria 594

We expected that MatR would be involved in the splicing of group II introns in the plant 595

mitochondria. To establish the roles of MatR in mitochondrial RNA metabolism, we used two 596

complementary approaches. The first strategy involved the identification of mt-RNAs that are 597

stably associated with MatR in vivo using RIP-chip assays (Schmitz-Linneweber et al., 2005; 598

20

Keene et al., 2006; Townley-Tilson et al., 2006). RNAs identified by the RIP-chip method 599

involved different transcripts that all contained group II introns, including nad1 i1, i3 and i4, 600

nad4 i1, nad5 i3 and i4, nad7 i2, rpl2 i1, and rps3 i1 (Figure 6 and Supplemental Figure 4). 601

Sequencing data from mt-RNAs, which co-precipitated with the MatR protein (Supplemental 602

File 1), indicated that the signals detected by the microarrays in the MatR co-IPs likely 603

corresponded to unspliced precursor RNAs, or that MatR is also associated with the spliced 604

exons, in vivo. Accordingly, the 3D structure of the ribonucleoprotein complex of LtrA bound to 605

its cognate ltrB intron RNA indicated that the spliced exons remain associated with the LtrA 606

protein, while the LtrA-depleted ltrB intron structures lack any exon sequences (Qu et al., 2016). 607

However, LtrA binds less tightly to the spliced ltrB intron RNA than to the exon-containing 608

precursor RNA, and the binding of LtrA to the intron lariat can be enhanced in the presence of 609

oligoribonucleotids, corresponding to sequences of intron-exon binding sites. The RIP-chip data 610

suggest that MATs in plant organelles also demonstrate high affinities in their binding to 611

precursor RNAs. 612

It was anticipated that MatR would associate in vivo with those introns whose splicing is 613

facilitated by the protein. However, this assumption needed to be supported experimentally. 614

Accordingly, the second approach involved a pioneering strategy for the down-regulation of the 615

expression of mitochondrial genes by synthetically designed ribozymes that are conjugated to a 616

tRNA-like shuttle vector RNA. Using a similar approach, the expression of the mitochondrial 617

atp9 mRNA and Atp9 subunit was reduced by about 80% and 50%, respectively (Val et al., 618

2011). As the functions of MatR were expected to be essential in the plant, the expression of the 619

matRz1 and matRz2 ribozymes (Figure 7) was regulated by the inducible β-estradiol-mediated 620

gene expression system (Zuo et al., 2000). 621

The expression of matRz1 and matRz2 ribozymes resulted in reductions in MatR gene 622

expression (up to 50% decrease in transcript levels and 20–50% reductions in MatR protein 623

levels in matRz2 and matRz1, respectively) (Figure 8 and Supplemental Figure 5), leading to 624

splicing deficiencies in many organellar pre-mRNAs in both transformant lines (Figure 9B). 625

These particularly included nad1 i3 and i4, as well as the splicing of efficiencies of nad5 i4, 626

nad7 i2, rpl2 i1 and rps3 i1 pre-RNAs (Figure 9B). A reduction in nad1 i3 pre-RNA may 627

correspond to an indirect effect, since the 3' part of this trans-spliced intron is co-transcribed 628

with nad1 i4 and the matR-ORF (Supplemental Figure 2). Thus, the cleavage of matR reading-629

21

frame may also lead to RNA instability of nad1 i3. On the other hand, the majority of the introns 630

whose splicing was affected by the expression of the matR ribozymes were also found to be 631

associated with MatR in vivo by the RIP-chip analysis (Figure 6 and Supplemental Figure 4), 632

including nad1 intron 3. It therefore anticipated that this intron, together with multiple other 633

group II introns highlighted in our experiments, is a processing substrate of MatR. Yet, some 634

pre-RNAs that were identified in the RIP-chip assay (Figure 6 and Supplemental Figure 4) were 635

not significantly affected in their splicing upon downregulating matR expression (i.e. nad1 i1 and 636

nad4 i1; Figure 9). Such differences between the RIP-chip data and the analysis of splicing 637

efficiencies in matR knockdown lines by RT-qPCR may correspond to the limited resolution of 638

the RNA footprints, but they more likely correspond to only a partial reduction in the expression 639

of MatR upon the induction of matRz1 or matRz2 ribozyme expression (see Figure 8 and 640

Supplemental Figure 5). Likewise, the nucleus-encoded nMAT1, nMAT2, and nMAT4 proteins 641

also facilitate the splicing of different subsets of mitochondrial introns (Keren et al., 2009; Keren 642

et al., 2012; Cohen et al., 2014), while the plastidial homolog, MatK, was shown to be associated 643

with many of the subgroup-IIA intron RNAs in vivo (Zoschke et al., 2010). Table 1 summarizes 644

the putative RNA targets of the MatR protein in Brassicales mitochondria. 645

646

MatR functions are likely to be critical during early stages in plant development 647

As indicated in Figure 8, splicing efficiencies were only partially affected in matRz1 and matRz2 648

lines, and significant levels of mature transcripts (mRNAs) corresponding to nad1, nad4, nad5, 649

nad7, cox2, ccmFc, rpl2, and rps3 were seen in both transgenic lines. It is possible that in 650

addition to MatR, the splicing of some of these mt-RNAs also involves additional nucleus-651

encoded factors (reviewed in (Brown et al., 2014; Schmitz-Linneweber et al., 2015). For 652

example, both nMAT4 and mCSF1 are involved in the maturation of nad1 i3, while the splicing 653

of nad1 i4 requires at least nMAT4 and MatR (Zmudjak et al., 2013; Brown et al., 2014; Cohen 654

et al., 2014; Schmitz-Linneweber et al., 2015). Yet, it is also anticipated that the RNA 655

phenotypes we observe correspond to only a partial silencing of MatR expression in the matRz 656

lines (Figure 8 and Supplemental Figure 5). We assume that the complete loss of MatR would 657

lead to far more profound defects in the processing of these RNAs, leading to embryo-lethal 658

developmental defects. Accordingly, while a complex I knockout is expected to be viable in 659

plants (Kuhn et al., 2015; Fromm et al., 2016; Ostersetzer-Biran, 2016), the functions of other 660

22

respiratory complexes, including CIII, CIV, and the translation machinery are considered 661

essential for normal embryo development (Gu et al., 1994; Berg et al., 2005; Meyer et al., 2005; 662

Colas des Francs-Small et al., 2012; Dahan et al., 2014). 663

While the splicing efficiencies of various transcripts were affected upon matR 664

knockdown, and their corresponding mRNA levels were reduced in the transformants, several 665

mt-mRNAs accumulated to rather higher levels in both the matRz1 and matRz2 lines (Figure 8B 666

and Supplemental Figure 5 and 7). These include transcripts corresponding to nad2 exons 3-4 667

and nad5 exons 1-2 in the matRz1 and matRz2 lines. An upregulation in the steady-state levels of 668

nad2 and nad5 RNAs in matR knockdown plants may correspond to the accumulation of 669

precursor transcripts that are correctly processed by various other splicing cofactors, but cannot 670

be spliced together with the other exons, of which the maturation relies on MatR. Analogously to 671

induced matRz1 and matRz2 lines, increases in the levels of various mRNAs were also apparent 672

in nmat1 (Keren et al., 2012) and nmat4 (Cohen et al., 2014) mutants, which are also affected in 673

the maturation of nad1 and show reduced complex I levels. In the matR knockdown lines, these 674

may involve small increases in levels of the ATP-synthase subunits atp6 (in both matRz1 and 675

matRz2 lines) and atp8 (only in matRz1, also annotated as orfB,) (Supplemental Figure 6). An 676

upregulation in the steady-state levels of mRNAs in mutants affected in mitochondrial RNA 677

metabolism and complex I biogenesis may correspond to compensatory effects due to the altered 678

organellar functions, involving reduced respiration and altered cellular metabolism (see e.g. 679

(Keren et al., 2012). We anticipate that a more profound effect on MatR expression would result 680

in notable changes in the levels of various other mt-RNAs, as seen in the cases of nmat1 and 681

nmat4 mutants. 682

683

Seed plants with unusual matR genes have also lost many group II introns from their 684

mitochondrial genomes 685

The matR ORF has been retained as a putatively functional gene within the nad1 i4 intron in the 686

mitochondrial genomes of numerous plant species, and until recently it was believed to be 687

invariantly present at this position in all seed plants. In fact, recent sequencing has demonstrated 688

that the matR gene can exist in the mtDNAs of several species (most Geraniaceae, the mistletoe 689

Viscum album, the gnetophyte Welwitschia mirabilis) as a freestanding gene (Park et al., 2015; 690

Petersen et al., 2015; Guo et al., 2016), while in some plants including Viscum scurruloideum, 691

23

Pelargonium x hortorum, Croizatia brevipetiolata, and Lachnostylis bilocularis, the matR gene 692

has been lost from their mtDNAs (Wurdack and Davis, 2009; Skippington et al., 2015) and 693

Supplemental Table 4. 694

Except for Croizatia and Lachnostylis, for which mitochondrial intron content is not 695

known, all of the other species with unusual matR genes have also experienced substantial loss of 696

their mitochondrial group II introns (see Supplemental Table 4). In V. scurruloideum¸ no nuclear 697

copy of matR was detected in the sequencing data (Skippington et al., 2015), and all 698

mitochondrial introns spliced by MatR have been lost, suggesting that MatR function has been 699

lost completely from this species. In P. hortorum, although the mitochondrial matR gene was lost 700

from the mtDNA, copies of the gene were found in the nuclear genome by DNA-seq analysis of 701

P. hortorum plants (Supplemental Table 4). Comparisons of the deduced amino acid sequences 702

between the nuclear genes and ‘canonical’ (i.e. mitochondria-encoded) MatR proteins suggest 703

that the original Ph.matR gene has been split in the nucleus into two isoforms, denoted here as 704

nMAT-X and nMAT-RT, both of which are predicted by their N-terminal sequences to be 705

targeted to the mitochondria (Figure 2). The nMATR-X isoform contains an intact domain X and 706

parts of the finger-palm RT sequence blocks (5-to-7), whereas the nMatR-RT isoform harbors 707

the upstream region with the remaining RT 2 to 4 motifs, which are conserved in the 708

mitochondria-encoded matR genes. In other Geraniaceae species, the matR gene was moved 709

from its ancestral position in the nad1 i4 intron to become a freestanding gene in the 710

mitochondrial genome. Although many introns were lost from the mitochondrial genomes of all 711

Geraniaceae species, the nad7 i2 gene is still present, consistent with a need for retained function 712

for MatR and thus explaining the retention of matR. It is also possible that MatR may function in 713

these species on additional intron targets. In W. mirabilis, numerous introns have been lost from 714

the mitochondrial genome, including most, but not all, of the splicing targets of MatR in 715

Brassicales. Here again, the matR gene is in an unusual configuration, but it has not been lost 716

from the genome due to the presence of nad1 i3, nad1 i4, and nad7 i2, which require MatR 717

activity to be spliced out (Supplemental Table 4). 718

Combined, these observations provide further evidence that the matR locus encodes a 719

functional protein, which is required for the processing of many group II introns. Only after all 720

intron targets of MatR have been lost can the matR gene be fully lost from the genome, as seen in 721

V. scurruloideum (Supplemental Table 4) (Skippington et al., 2015). The V. album mitochondrial 722

24

genome has also lost all introns that require MatR activity for removal, yet a nearly complete 723

matR gene has still been retained. However, the 3’ truncation of about 150 bp suggests that the 724

gene may be in the initial stages of degradation, and it is possible that the gene is no longer 725

transcribed, although transcriptional activity was not assayed in the previous study (Petersen et al 726

2015). In Geraniaceae and Welwitschia, the loss of most introns processed by MatR correlates 727

with a modified configuration for the matR gene. These findings suggest that the loss of several 728

intron targets may reduce functional constraints on localization or co-transcription with the nad1 729

gene, enabling matR to persist as a freestanding gene in the mitochondrial or nuclear genome and 730

be transcribed independently from nad1. Nevertheless, because at least one intron spliced out by 731

MatR remains, the matR gene has still been retained in some form. These scenarios could signify 732

an intermediate stage of evolution where matR activity is reduced and may eventually be lost 733

upon loss of the remaining matR-processed introns. 734

735

MatR: an evolutionary step between the highly ‘specialized’ bacteria maturases and the 736

general spliceosomal machinery in eukaryotes? 737

Group II introns are catalytic RNAs that exist in the genetic systems of the three major domains 738

of life (i.e. archaea, bacteria and eukaryotes). Based on structural features, the similarities of 739

exon-intron boundaries and a common splicing mechanism, group-II introns are proposed to be 740

the progenitors of the eukaryotic spliceosome system (see e.g. (Schmitz-Linneweber et al., 741

2015). It has been postulated that group II introns invaded the nuclear genomes of eukaryotes 742

early during the evolutionary transition from an (α-proteo)-bacterial endosymbiont to the 743

mitochondrion. In support of this idea, some bacterial maturases have the ability to support 744

retrohoming, and recent studies have shown homology between group II maturases and the core 745

spliceosomal Prp8 factor (Dlakic and Mushegian, 2011). The structures of bacterial maturases 746

bound to their host RNAs (Qu et al., 2016; Zhao and Pyle, 2016) further support this intriguing 747

theory. Yet, it still remains unclear how the universal spliceosomal splicing machinery evolved 748

from the bacterial group II introns and their related monospecific maturase factors. Throughout 749

their evolution, organellar introns in plants have diverged considerably from their self-splicing 750

group II RNAs (Bonen, 2008). This divergence in sequence and structure has been accompanied 751

by the acquisition of a large number of accessory protein cofactors. All of the plant maturases 752

(i.e. MatR, MatK and the nMATs) act on multiple pre-RNA targets and function in the splicing 753

25

of group II RNAs. Also required are a number of additional (nucleus-encoded) splicing 754

cofactors, possibly acting as an ‘organellar proto-spliceosome’ (Schmitz-Linneweber et al., 755

2015). It is possible that in parallel to the invasion of group II introns into the nucleus of 756

eukaryotes, a general maturase has evolved early during evolution to function in the splicing of 757

many RNA targets in a similar manner to the acquisition of several organellar intron targets by 758

the organellar maturases in plants. 759

Our hypothesis is that the MatR protein is found in the mitochondria in ribonucleoprotein 760

(RNP) particles containing different group II introns and various splicing cofactors. The 761

generalization of the intron targets of MatR makes this protein an interesting model for the 762

evolution of the nuclear spliceosomal machinery, which also underwent a process of splicing-763

target generalization. Interestingly, MatR lacks the N-terminal domain (NTD), which in the case 764

of the bacterial LtrA is required for its association with the DIV domain of the ltrB intron (or 765

possibly to control its own translation, in vivo) (Qu et al., 2016), and also harbors two amino 766

acids sequence insertions within the figure-palm region (Figure 1B). The lack of a NTD 767

subdomain and the presence of additional motifs within the RT (i.e. finger-palm) domain may 768

coincide with the loss of a specific target in parallel to the acquisition of multiple intron RNA 769

targets. Likewise, the Prp8 protein also harbors additional sequence blocks within the fingers-770

palm region of the RT domain, which function in intron binding (Galej et al., 2013; Yan et al., 771

2015; Qu et al., 2016). 772

773

METHODS 774

Plant material and growth conditions 775

Arabidopsis thaliana ecotype Columbia (Col-0) seeds were obtained from the ARBC at Ohio 776

State University (Columbus, OH). Prior to germination, seeds of wild-type or matRz/sdhRz 777

transformants were surface-sterilized with bleach (sodium hypochlorite) solution and sown on 778

MS-agar plates containing 1% (w/v) sucrose. The plates were kept in the dark for a few days at 779

4°C and then transferred to controlled temperature and light conditions in growth chambers. 780

Wild-type plants were grown in a controlled environment plant growth chamber (Arabidopsis 781

Chamber AR-41L3, Percival Scientific Inc. Perry, IA) at 22°C with 50% relative humidity, under 782

short day conditions (i.e. 16 hour dark and 8 hour light), at approximately 150 µE·m-2·s-1 s 783

26

(PHILIPS bulbs type F17T8/L841). Cauliflower (Brassica oleracea var. botrytis) inflorescences 784

were purchased fresh at local markets. 785

786

Gene constructs for ribozyme-mediated knockdown of matR 787

Constructs for the expression of the matRz1, matRz2 and sdhRz ribozymes associated with the 788

PKTLS shuttle were prepared by PCR amplification with the large direct primers 789

dirRz1matrXho, dirRz3matrXho and dirRzsdhXho, respectively (Supplemental Table 3). These 790

primers included, from 5' to 3', an Xho I site, the complete sequence of the relevant ribozyme, 791

the linker sequence and nucleotides 100-120 from the 3'-end of the Turnip yellow mosaic virus 792

(TYMV) genomic sequence. The revHDVSpe reverse primer (Supplemental Table 3B) included, 793

from 5' to 3', an SpeI site and the reverse complement to nucleotides 1 to 20 from the 3'-end of 794

the Hepatitis delta virus antigenomic cis-cleaving ribozyme (cHDV) (Perrotta and Been, 1991). 795

The pBI-PSTYPKTLScHDV plasmid (Val et al., 2011), containing the last 186 nucleotides from 796

the 3'-end of the TYMV genomic sequence fused to the sequence of the cHDV cis-ribozyme, 797

was used as a template. The resulting PCR products, encoding the trans-ribozymes combined 798

with the linker, the PKTLS and the cHDV cis-ribozyme, were directly cloned into the pGEM-T 799

vector (Promega) for sequencing, re-excised with the XhoI and SpeI restriction endonucleases 800

and cloned into the XhoI and SpeI sites of the estradiol-inducible transcription unit of the pER8 801

vector (Zuo et al., 2000). Transcription of these constructs by RNA polymerase II yields 5'-802

capped transcripts comprising the matRz1, matRz2 or sdhRz ribozyme, the linker, the PKTLS, 803

the cHDV, the termination sequence and a poly(A) tail. Self-cleavage of the cHDV eliminates 804

the termination sequence and the poly(A) tail and releases the regular 3'-CCA end of the PKTLS. 805

The resulting ribozyme-linker-PKTLS RNA is exported from the nucleus to the cytosol, 806

recognized by the mitochondrial tRNA import machinery and imported into the organelles (Val 807

et al., 2011). 808

809

Nuclear transformation and chimeric ribozyme expression 810

The recombinant pER8 plasmids carrying the ribozyme-linker-PKTLS-cHDV constructs (Figure 811

7 and Supplemental Table 3B) served to transform A. thaliana (Col-0) plants via A. tumefaciens 812

through floral dip (Clough and Bent, 1998). Primary transformants were selected on solid agar 813

medium containing hygromycin. Resistant plants were grown in the greenhouse to the next 814

27

generations and homozygous transformants were selected for further studies. To induce 815

ribozyme expression and matR mRNA knockdown, seedlings were grown on solid agar medium 816

in complete darkness or in long daylight conditions (16 hour light/8 hour dark). In complete dark 817

conditions, the agar medium was overlaid with 5 μM β-estradiol-supplemented liquid medium 818

for transgene induction. In light conditions, the seedlings were transferred to 6-well culture 819

plates containing liquid medium supplemented with estradiol, with the roots dipping into the 820

medium. The matRz1 and matRz2 lines represent two individual transformant lines, expressing 821

the trans-ribozymes designed to specifically cleave the Arabidopsis matR transcript 822

(ArthMp045) in different gene-loci (see Figure 7). The sdh3 line expresses a ribozyme (sdhRz) 823

designed against the mitochondrial sdh3 in Nicotiana tabacum (NitaMp030), which was used as 824

a control for the matR knockdown experiments, since it has no corresponding targets in the 825

Arabidopsis mtDNA (NC_001284). ‘Positive’ transformant lines (i.e. sdh3, matRz1 and matRz2) 826

were further screened based on the lack of obvious phenotypes in the un-induced state, while 827

showing a notable expression levels upon estradiol induction. For the analysis of matR 828

knockdown effects on mitochondrial gene-expression and biogenesis, we selected matRz plants, 829

which showed equivalent levels of transgene (i.e. matRz1 or matRz2) expression to those seen for 830

the sdhRz construct in the sdh3 line. Full biological replicates (i.e., separate experiments) were 831

then run with each selected transformant. We used a transient induction (i.e. 5 ~ 6 days) for the 832

expression of each ribozyme transgene, as estradiol may be metabolized by the plant cells 833

(Janeczko and Skoczowski, 2005). 834

835

RNA extraction and analysis 836

RNA extraction was performed essentially as described previously (Keren et al., 2011; Cohen et 837

al., 2014). In brief, RNA was prepared following standard TRI Reagent protocols (Molecular 838

Research Center) with additional phenol/chloroform extraction. The RNA was treated with 839

RNase-free DNase (Ambion) prior to its use in the assays. RT-qPCR was performed with 840

specific oligonucleotides designed to intron-exon regions (pre-mRNAs) and exon-exon 841

(mRNAs) regions corresponding to the 23 intron-containing mitochondrial transcripts in wild-842

type and matR knockdown (matRz) plants, generally as previously described (Koprivova et al., 843

2010; Kühn et al., 2011). Reverse transcription was carried out with the Superscript III reverse 844

transcriptase (Invitrogen), using 1 - 2 µg of total RNA and 250 ng of a random hexanucleotide 845

28

mixture (Promega) and incubated for 50 min at 50oC. Reactions were stopped by 15 min 846

incubation at 70°C and the RT samples served directly for PCR. Quantitative PCR (qPCR) 847

reactions were run on a LightCycler 480 (Roche), using 2.5 μL of LightCycler 480 SYBR Green 848

I Master mix and 2.5 μM forward and reverse primers in a final volume of 5 µL. Reactions were 849

performed in triplicate in the following conditions: pre-heating at 95°C for 10 min, followed by 850

40 cycles of 10 sec at 95°C, 10 sec at 58°C and 10 sec at 72°C. For the analysis of MatR 851

expression, the nuclear genes ACTIN 2, GAPDH and EXPRESSED were taken as reference 852

genes (Czechowski et al., 2005). For the analysis of mitochondrial gene expression and RNA 853

accumulation, the nucleus-encoded 18S rRNA subunit and the mitochondrial 26S rRNA (rrn26) 854

subunit were used as reference genes in the qPCR analyses (Koprivova et al., 2010; Kühn et al., 855

2011). 856

857

Transcriptome mapping by high-throughput (RNA-seq) analysis 858

Total mt-RNA was extracted from highly enriched mitochondria preparations obtained from 859

Brassica oleracea var. botrytis (cauliflower) inflorescence (see below). Sequencing was carried 860

out on an Illumina Genome Analyzer (The Genome High-Throughput Sequencing Laboratory, 861

Tel-Aviv University, Israel), to generate transcriptome profiles and evaluated by mapping of the 862

B. oleracea mt-RNA sequences onto the genomic reference sequence using bowtie 2.1.0 with 863

default parameters, essentially as described by (Grewe et al., 2014). To reduce mapping of 864

chloroplast transcript reads to MIPT (mitochondrial DNA of plastid origin) regions or putative 865

paralogous mitochondrial genes (e.g. plastid atpA and mitochondrial atp1), we added the 866

Brassica napus chloroplast genome sequence (NC_016735) to the mitochondrial reference 867

sequence as bait for native plastid sequences. Bowtie results in SAM format were aligned using 868

utilities from SAMtools (Li et al., 2009). A custom Perl script was used to determine the depth 869

and direction of transcription at each nucleotide in the reference genome. The expression level of 870

each gene was determined by the average of transcript coverage at each nucleotide. 871

872

Generation of rabbit polyclonal antibodies against the MatR protein 873

Since no antibodies to MatR protein were available prior to this study, a polyclonal antibody was 874

raised against a synthetic peptide corresponding to a unique region in MatR’s RT domain (i.e. 875

29

NH3-RRIDDQENPGEEASFNA), which is conserved among different Brassicales (see 876

Supplemental Figure 1). The antibody was purified from the serum by affinity chromatography 877

column and was then tested for its ability to recognize the immunizing peptide. The affinity and 878

specificity of the purified MatR antibody were further evaluated in protein gel-blots, using total 879

protein and enriched organelle preparations (i.e. mitochondria, chloroplasts and peroxisomes) 880

obtained from 3-week-old Arabidopsis thaliana var. Columbia and purified mitochondria 881

extracted from cauliflower inflorescences. 882

883

RIP-chip assays 884

Mitochondrial preparations from cauliflower inflorescences were solubilized with 1% NP-40 885

(v/v) in assay buffer (150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2) at a protein 886

concentration of about 1 mg/mL. After 30 min incubation on ice, the organellar extract was 887

centrifuged for 10 min at 21,000 g. The clear supernatant was then incubated with affinity-888

purified anti-MatR antibodies conjugated to protein A/G Sepharose beads (Amersham), with 889

gentle mixing at 4oC for 16 hr. The beads were collected by a brief centrifugation (1 min at 1,000 890

g, at 4oC) and washed three times with 0.6 M NaCl, 0.5% (v/v) NP-40, 50 mM Tris-HCl (pH 891

8.3), followed by a single wash with 1x PBS buffer. Dot-blot analysis of co-immunoprecipitated 892

RNA was performed essentially as described in (Zoschke et al., 2010). Total mt-RNA co-893

precipitated with the anti-MatR antibodies was DNase digested and then reverse transcribed 894

using Superscript III reverse transcriptase (Invitrogen) and a random hexanucleotide mixture 895

(Promega) in the presence of [α32P]-dCTP and [α32P]-dATP (to increase specific signals). The 896

identity of the proteins in the co-IPs was established by LC-MS/MS analysis (Smoler Proteomics 897

Center, Technion, Israel). Commercial antibodies to SHMT were used as controls for the 898

integrity of the RIP-chip method. 899

900

Mitochondria extraction and analysis 901

Preparations of highly enriched mitochondria from cauliflower inflorescence generally followed 902

the protocol described in detail in (Neuwirt et al., 2005). About 1.0 kg fresh weight 903

inflorescences (two to three heads of cauliflower) were cut off from the stems and kept overnight 904

(about 16 hours) in cold water (4oC). On the following morning, the inflorescences were ground 905

with ice-cold extraction buffer [0.9 M mannitol, 90 mM Na-pyrophosphate, 6 mM EDTA, 2.4% 906

30

PVP25 (w/v), 0.9% BSA (w/v), 9 mM cysteine, 15 mM glycine, and 6 mM β-mercaptoethanol; 907

pH 7.5]. Mitochondria were recovered from the extract by differential centrifugations and 908

purification on Percoll gradients. Following fractionation, the mitochondria were pelleted, 909

resuspended in a small volume of wash buffer (0.3 M mannitol, 10 mM K-phosphate, 1 mM 910

EDTA, pH 7.5), aliquoted and stored frozen at -80oC. For the isolation of mitochondria from 911

Arabidopsis, we used 2-week-old seedlings grown on MS-plates. The enriched organelle 912

preparation was performed essentially as described in (Keren et al., 2012). 913

914

Mitochondrial sub-fractionation into matrix and membranes 915

Intact mitochondria were disrupted by several freeze–thaw cycles in wash buffer (i.e. 0.3 M 916

mannitol, 10 mM KH2PO4, 1 mM EDTA, pH 7.5), briefly sonicated and then centrifuged at 917

25,000 g for 20 min to separate the matrix (supernatant) and membrane (pellet) fractions. Wash 918

buffer, equal to the initial volume, was added to the pellet and briefly sonicated. Whole 919

mitochondria and the organellar subfractions were diluted in 1:1 ratio with 3X Laemmli sample 920

loading buffer (Laemmli, 1970) supplemented with 50 mM β-mercaptoethanol, heated at 70°C 921

for 10 min and cleared by centrifugation at 25,000 g for 5 min. To determine whether 922

membranous proteins are peripherally or integrally bound to the membranes, the membrane 923

fraction (equ. of 40 mg leaves) was pre-washed with 200 µl 10 mM Tris-HCl pH 8.0 (for the 924

removal of contaminating proteins) and pelleted by centrifugation for 8 min at 21,000 g (4oC). 925

The membranes were then treated with either 1M NaCl (removes loosely attached membrane 926

proteins), 2M NaBr (a chaotropic agent used for the removal of peripherally-bound proteins), or 927

0.05% Triton X-100, 1M NaCl (wash some peripheral proteins attached by surface hydrophobic 928

interactions), generally as described in (Ostersetzer et al., 2007). The location of each protein 929

(i.e. peripherally or integrally associated) was determined by centrifugation (i.e. 8 min at 21,000 930

g, 4oC) and immunoblot analysis of both the pellet (containing the membranes) and supernatant 931

fractions. 932

933

Protein extraction and analysis 934

Protein extraction and analysis were performed generally as described in (Keren et al., 2009). 935

Total protein was extracted from 3-week-old Arabidopsis leaves or isolated mitochondria 936

31

obtained from cauliflower inflorescences by the borate/ammonium acetate method (Maayan et 937

al., 2008). For this purpose, frozen plant tissue was homogenized in the presence of 938

Polyvinylpolypyrrolidone (PVPP). The homogenate was added to microfuge tubes containing 939

400 mL ice-cold protein extraction buffer [50 mM Na-borate, 50 mM ascorbic acid, 1.25% (w/v) 940

sodium dodecyl sulfate (SDS), 12.5 mM b-mercaptoethanol, pH 9.0] and the protease inhibitor 941

cocktail ‘complete Mini’ from Roche Diagnostics GmbH (Mannheim, Germany). Proteins were 942

recovered by centrifugation (25,000 g) in the presence of three volumes of ice-cold 0.1 M 943

ammonium acetate in methanol buffer (NH4-OAc-MeOH), generally as described in (Maayan et 944

al., 2008). Protein concentration was determined according to the Bradford method (BioRad), 945

with bovine serum albumin used as a standard. Approximately 20 µg total protein was mixed 946

with an equal volume of 3X protein sample buffer (Laemmli, 1970), supplemented with 50 mM 947

β -mercaptoethanol, and subjected to 12% SDS-PAGE (at a constant 100 V). Following 948

electrophoresis, the proteins were transferred to a PVDF membrane (BioRad) and blotted 949

overnight at 4°C with specific primary antibodies. Detection was carried out by 950

chemiluminescence assays after incubation with an appropriate horseradish peroxidase (HRP)-951

conjugated secondary antibody (Sigma or Santa Cruz). 952

For matRz and sdhRz Arabidopsis transformants, plant samples were weighed, snap 953

frozen in liquid nitrogen and ground in liquid nitrogen with pre-chilled mortar and pestle. The 954

fine frozen powder was transferred to a 1.5 mL microtube and 10 µL extraction buffer [140 mM 955

Tris base, 105 mM Tris-HCl pH 7.0, 0.5 mM EDTA, 2% (w/v) SDS, 10% (v/v) glycerol, 0.1 956

mg/mL protease inhibitors (Roche)] was added per mg sample wet weight. The suspension was 957

immediately re-frozen in liquid nitrogen and sonicated until it was just thawed. The procedure 958

was repeated 3 times with 1 min breaks in liquid nitrogen. The samples were subsequently 959

centrifuged for 3 min at 10,000 g to remove insoluble material and the supernatants were used 960

for protein gel blot analysis. Proteins (50 μg and 5 μ g for MatR and β -actin detection, 961

respectively) were separated on 12.5% (w/v) SDS-PAGE gels and blotted onto PVDF 962

membranes. Immunodetection was carried out with a SNAP 2.0 Protein Detection System 963

(Merck Milllipore), according to the manufacturer’s instructions. MatR was immunoprobed with 964

a polyclonal rabbit antiserum raised against the overexpressed and affinity-purified N-terminal 965

half of the protein. β-actin was detected with a commercial mouse monoclonal antibody (Actin 966

32

MA1-744, ThermoFisher Scientific). Bound specific antibodies were revealed with biotinylated 967

polyclonal anti-rabbit IgG or monoclonal anti-mouse IgG secondary antibodies (Sigma), 968

streptavidin-alkaline phosphatase conjugate (Amersham Biosciences) and FASTTM BCIP/NBT 969

buffer (Sigma). Immunoblot signals were quantified with the MultiGauge software (Fujifilm). 970

971

Seed protein extraction and analysis 972

Total seed protein extracts were prepared from dry mature and viable seeds at different stages of 973

imbibition and germination. About 100 mg Arabidopsis seed [~5,000 seeds; (Meinke, 1994)] 974

was ground with mortar and pestle in the presence of 2 ml extraction buffer [75 mM MOPS-975

KOH, 0.6 M Sucrose, 4 mM EDTA, 0.2% PVP-40, 0.2% BSA, 8 mM L-cystein, pH 7.6] and the 976

protease inhibitor cocktail ‘complete Mini’ from Roche Diagnostics GmbH (Mannheim, 977

Germany). Crude membrane extracts were prepared essentially as described in (Colas des 978

Francs-Small et al., 2012). The membranous fraction was obtained by centrifugation at 22,000 g 979

for 10 min at 4oC. The pellet containing the crude membranous fraction was washed twice with 980

wash buffer [37.5 mM MOPS-KOH, 0.3 M Sucrose, 2 mM EDTA pH 7.6]. The samples were 981

kept frozen at -80°C until used in denaturing (i.e. SDS-PAGE) or Blue native (BN) gel 982

electrophoresis. 983

984

Blue native (BN) electrophoresis for isolation of native organellar complexes 985

Blue native (BN)-PAGE of crude membranous fractions was performed according to the method 986

described by (Zmudjak et al., 2013). An aliquot equivalent to 25 mg of crude membrane extracts, 987

obtained from Arabidopsis thaliana (Col-0) seeds or leaves from 3-week-old seedlings, was 988

solubilized with n-dodecyl-ß-maltoside (1.5% [w/v]) in ACA buffer (750 mM amino-caproic 989

acid, 0.5 mM EDTA, and 50 mM Tris-HCl, pH 7.0), and then incubated on ice for 30 min. The 990

samples were centrifuged 8 min at 20,000 g, and Serva Blue G (0.2% [v/v]) was added to the 991

supernatant. The samples were then loaded onto a native 4 to 16% gradient gel. For non-992

denaturing-PAGE-protein gel blotting, the gel was transferred to a PVDF membrane (Bio-Rad) 993

in Cathode buffer (50 mM Tricine and 15 mM Bis-Tris-HCl, pH 7.0) for 16 h at 4°C (const. 40 994

mA). The membrane was incubated with various antibodies, as indicated in each blot, and 995

detection was carried out by chemiluminescence assay after incubation with an appropriate 996

horseradish peroxidase (HRP)-conjugated secondary antibody. 997

33

998

Accession numbers 999

Sequence data from this article can be found in the GenBank/EMBL libraries under accession 1000

numbers: Arabidopsis thaliana mitochondria genome sequence (NC_001284); Brassica oleracea 1001

var. botrytis mitochondria genome sequence (KJ820683); Arabidopsis ACTIN2 (At3g18780); 1002

Arabidopsis atp6 (ArthMp035); Arabidopsis atp8 (also annotated as orfB, ArthMp040); 1003

Arabidopsis EXPRESSED (At4g26410); Arabidopsis GADPH (At1g13440); Arabidopsis matR 1004

(ArthMp045); Arabidopsis mitochondrial rRNA subunit 18S protein (At3g41768); Arabidopsis 1005

mitochondrial 26S rRNA (rrn26) subunit (ArthMr001); Nicotiana tabacum sdh3 (NitaMp030); 1006

Turnip yellow mosaic virus (TYMV) genome sequence (X16378). 1007

1008

Supplemental Data 1009

Supplemental Figure 1. Alignment of MatR homologs from various angiosperms. 1010

Supplemental Figure 2. Transcription profile of the nad1 ex4-int4-ex5 gene locus in 1011

cauliflower mitochondria according to RNA-seq analysis. 1012

Supplemental Figure 3. Analysis of matR transcription in Brassicaceae. 1013

Supplemental Figure 4. Identification of MatR-associated mitochondrial RNAs by dot-blot 1014

hybridizations. 1015

Supplemental Figure 5. Expression of intronless mitochondrial mRNAs in matR knockdown 1016

lines. 1017

Supplemental Figure 6. Phenotype of Arabidopsis thaliana transgenic lines expressing the 1018

matRz ribozymes. 1019

Supplemental Figure 7. Accumulation of mitochondrial mRNAs in matR knockdown lines. 1020

Supplemental Figure 8. Homology modeling of the Arabidopsis thaliana maturase related 1021

(MatR) protein. 1022

Supplemental Table 1. List of antibodies used for the analysis of MatR. 1023

Supplemental Table 2. Proteins identified by LC/MS-MS analysis in the RIP-chip assays. 1024

Supplemental Table 3. Lists of oligonucleotides. 1025

Supplemental Table 4. The matR gene status in different angiosperms. 1026

Supplemental File 1. Identification of MatR-associated mitochondrial RNAs by RNA-co-1027

immunoprecipitation/cDNA sequencing. 1028

34

1029

ACKNOWLEDGMENTS 1030

We thank Prof. Christian Schmitz-Linneweber (Humboldt University, Berlin) for help with 1031

mitochondria RNA-seq data and dot-blot analysis of RNA co-IPs, Prof. Ian Small (University of 1032

Western Australia) for the assistance with RT-qPCR analyses, Dr. Yoram Eyal (The Volcani 1033

research Inst.) for providing us with the RACE protocols, Amalia Biran-Ostersetzer for the 1034

assistance with mitochondria RNA-seq analysis, Dr. Maciej Szymanski (A. Mickiewicz 1035

University, Poznan) for help in ribozyme design, Dr. Marta Gabryelska (PAN-IBCH, Poznan) 1036

for help in ribozyme testing, Engr. Anne Cosset for help in transformant analysis and Dr. Sam 1037

Aldrin for his careful reading of the manuscript and points raised. This work was supported by 1038

grants to OOB from the Israeli Science Foundation (No. 741/15) and in part from the German-1039

Israeli Foundation (GIF 1213/2012), to AD from the French State program "Investments for the 1040

future" (LABEX ANR-11-LABX-0057_MITOCROSS) and from the French National Research 1041

Agency (ANR-06-MRAR-037-02, ANR-09-BLAN-0240-01), as well as a grant to JB from the 1042

Polish National Science Centre (UMO-2013/09/B/NZ1/03359) and to JPM from the US National 1043

Science Foundation (IOS-1027529 and MCB-1125386). Regular funding to AD from the French 1044

National Center for Scientific Research (CNRS-UPR2357) and the University of Strasbourg is 1045

acknowledged. 1046

1047

AUTHOR CONTRIBUTIONS 1048

L.S. did most of the biochemical analysis of MatR expression and performed co-IPs experiments, 1049

analysis of the RNA profiles of cauliflower and Arabidopsis mitochondria and the analysis of 1050

matR-knockdown lines by RT-qPCR. D.M. was involved in the establishment of matRz 1051

transgenic lines and analysis of the mt-RNA profiles of matR-knockdown lines. F.G. assisted 1052

with the sequencing and analysis of plant mitochondrial genomes and preformed bioinformatics 1053

analysis of mitochondria transcriptomes, intron distribution in angiosperms mtDNAs, and MatR 1054

status in plants. K.R. assisted with the RNA and protein analyses of matR-knockdown lines. S.A. 1055

produced antibodies to MatR and assisted in theRIP-chip analyses of cauliflower mitochondria 1056

MatR protein. P.G. did RNA and RT-qPCR analyses of matR-knockdown lines. A.K.N. 1057

participated in the development of the sdhRz ribozyme system in Arabidopsis and tobacco. I.K. 1058

assisted with the RIP-chip assays and performed hybridization assays of the RIP-chips. S.F. 1059

35

assisted with plant growth and analysis, protein and RNA extraction and analysis. L.K. assisted 1060

with the 3D modeling of MatR structures. J.B. and A.D. were involved in the development of the 1061

mitochondrial ‘ribozyme system’. J.B., J.P.M., A.D. and O.O.B. supervised the study. O.O.B. 1062

finalized the manuscript. 1063

1064

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1447 1448 Table 1. List of identified group II intron targets of MatR protein in Brassicales 1449

1450

Gene / Intron configuration chips-RIP matRknockdowns

matR

i1 rpl2 cis + +

i1 rps3 cis + +

i1 cox2 cis - +

i1 ccmFc cis - +

i1 nad1 trans + -

44

i2 nad1 cis (1)(+) -

i3 nad1 trans + +

i4 nad1 cis + +

i1 nad2 cis (+) -

i2 nad2 trans (+) -

i3 nad2 cis - -

i4 nad2 cis - (+)

i1 nad4 cis (+) -

i2 nad4 cis (+) -

i3 nad4 cis - (+)

i1 nad5 cis - -

i2 nad5 trans - -

i3 nad5 trans + (+)

i4 nad5 cis + +

i1 nad7 cis - -

i2 nad7 cis + +

i3 nad7 cis - -

i4 nad7 cis - -

1451 1452

1 Brackets indicate the data that could not be fully supported by the RIP-chip assays or the matR-1453 knockdown experiments. 1454

DOI 10.1105/tpc.16.00398; originally published online October 19, 2016;Plant Cell

Dietrich and Oren OstersetzerAdnan Khan Niazi, Ido keren, Sofia Shevtsov, Liron Klipcan, Jan Barciszewski, Jeffrey P Mower, Andre Laure D Sultan, Daria Mileshina, Felix Grewe, Katarzyna Rolle, Sivan Abudraham, Pawel Glodowicz,

II introns in Brassicaceae mitochondriaThe reverse-transcriptase/RNA-maturase protein MatR is required for the splicing of various group

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