The pre-mRNA Splicing Machinery of Trypanosomes: Co mplex ... · 25.06.2010 · Both types of...

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The pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?

Arthur Günzl

Department of Genetics and Developmental Biology and Department of Molecular, Microbial and

Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT

06030-3301, USA

Dr. Arthur Günzl

Department of Genetics and Developmental Biology

University of Connecticut Health Center

263 Farmington Avenue

Farmington, CT 06030-3301

USA

Phone (860) 679-8878

Fax (860) 679-8345

E-mail [email protected]

Short Title: pre-mRNA splicing in trypanosomes

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00113-10 EC Accepts, published online ahead of print on 25 June 2010

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ABSTRACT

Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei,

Trypanosoma cruzi, and several species of Leishmania cause severe, often lethal diseases in humans.

To better combat these parasites, their molecular biology has been a research focus for more than

three decades and the discovery of spliced leader (SL) trans splicing in T. brucei established a key

difference between parasites and hosts. In SL trans splicing, the capped 5' terminal region of the

small nuclear SL RNA is fused onto the 5' end of each mRNA. This process, in conjunction with

polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional

mRNA by providing the cap structure. The reaction is a two step transesterification process

analogous to intron removal by cis splicing which, in trypanosomatids, is confined to very few pre-

mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome consisting of five U-

rich small nuclear (sn)RNAs and, in humans, of up to ~170 different proteins. While

trypanosomatids possess a full set of spliceosomal U snRNAs, only few splicing factors were

identified by standard genome annotation because trypanosomatid amino acid sequences are

among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made

in the characterization of the splicing factor repertoire in T. brucei which was achieved by tandem

affinity purification of splicing complexes, by systematic analysis of proteins containing RNA

recognition motifs, and by mining the genome database. In addition, recent findings about

functional differences between trypanosome and human pre-mRNA splicing factors are discussed.


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Trypanosomatids are protistan parasites infecting hosts as diverse as mammals, insects and plants. In

humans, vector-borne Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. cause lethal

diseases and the strong impact of these parasites on global health has spurred investigations of the

molecular processes in these organisms early on. One of the first key discoveries in regard to gene

expression was spliced leader (SL) trans splicing which was eventually found to be an essential

maturation step for all nuclear pre-mRNA in trypanosomatids.

The initial discoveries of SL trans splicing were made in T. brucei and until now this organism has

remained the preferred trypanosomatid organism for spliceosomal studies. T. brucei is an extracellular

parasite which evades the mammalian immune system by antigenic variation of its variant surface

glycoprotein (VSG) coat. VSG expression has therefore been a research focus and it was on VSG mRNAs

that the 5' terminal region was first discovered to contain a leader sequence which was not encoded in the

VSG gene (11, 87). Further analysis showed that the 39 nt-long leader was derived from the 5' terminus of

a separate, small nuclear RNA, which has been termed SL RNA or mini-exon-derived RNA (13, 35, 57).

Discovery of a Y structure intermediate, which corresponds to the cis splicing intron-exon-lariat structure

(Fig. 1), strongly indicated that the SL transfer functions analogously to intron removal entailing the same

two transesterification reactions (59, 80). This notion was confirmed by the demonstration that destruction

of spliceosomal Uridine-rich small nuclear RNAs (U snRNAs) blocked SL transfer (85).

SL trans splicing is not restricted to VSG mRNA but is an essential maturation step for all

trypanosomatid mRNAs. In trypanosomatid genomes, coding genes are tandemly arranged in large

polygenic clusters which are transcribed in a polycistronic fashion (8). Trans splicing and polyadenylation

lead to precursor cleavages up- and downstream of a coding region, respectively, and therefore are

mechanistically required to process individual mRNAs from polycistronic pre-mRNA. Moreover, the SL

carries an m7G cap and the first four nucleotides of its sequence are methylated; some of these


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methylations are unique to trypanosomes and the unusual 5' terminal structure has been termed cap 4 (5).

Since cap 4 is transferred onto mRNA 5' ends as part of the SL, trans splicing represents a post-

transcriptional mode of mRNA capping and, therefore, is essential in the formation of functional mRNA.

Since all trypanosomatid mRNAs are trans spliced and trypanosomatid genes typically do not harbor

introns, it was long thought that these organisms use RNA splicing exclusively for SL transfer and

accordingly, trypanosome-specific deviations of splicing factors were hypothesized to be trans splicing-

specific. It therefore came as a surprise when the T. brucei PAP gene (Access. No. Tb927.3.3160)

encoding Poly(A) polymerase was shown to harbor a single intron that was removed by conventional cis

splicing (49). The search for further introns revealed only one more gene in T. brucei (Tb927.8.1510),

encoding a putative RNA helicase (8), whose pre-mRNA was shown to be cis spliced (31). Interestingly,

a recent characterization of the T. brucei transcriptome by RNA-seq strongly indicates that there are no

other introns disrupting protein coding genes (76).

SL trans splicing is a more widespread phenomenon in eukaryotes and after its initial discovery in

trypanosomes, it was found to occur in a variety of organisms including euglenids (81), nematodes (36),

trematodes (71), and even lower chordates such as the sea squirt (89). However, there is no indication that

this particular mode of trans splicing occurs in hosts of trypanosomatid parasites, e.g. vertebrates or

arthropods (20), and therefore it can be regarded as a parasite-specific process. This specificity and the

ubiquitous requirement of SL trans splicing for mRNA maturation has made this process an attractive

research focus. Long-term aims have been to find out how the trypanosome splicing machinery differs

from its human counterpart, to identify factors or factor domains which are specifically required for the

trans splicing process, and to analyze whether these features can be inactivated in a parasite-specific

manner. The challenge of this research is that the splicing machinery, termed spliceosome, is a huge,

dynamic complex composed of structural RNAs and proteins that is difficult to characterize.


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The spliceosome consists of the U1, U2, U4, U5, and U6 snRNAs and, in the human system, of up to

170 spliceosome-associated protein factors (92). Trypanosomatids possess all five spliceosomal U

snRNAs which are typically somewhat smaller and deviate in several aspects from their human

counterparts. In contrast, until recently, our knowledge of spliceosomal protein factors in trypanosomatids

was very limited. A main reason for this lack of knowledge comes from the fact that amino acid

sequences of trypanosomatid proteins have diverged dramatically from their human and yeast orthologs

and thus, only a few splicing factors were identified by standard annotation of the completed TriTryp

genomes (30). In the past years, however, major progress has been made in the identification of

spliceosomal proteins and the characterization of U small nuclear ribonucleoprotein particles (U snRNPs)

in trypanosomes. Three factors have contributed to this success: firstly, the unrestricted access to the

sequenced and annotated TriTryp genome databases (8) at GeneDB (http://www.genedb.org/) and,

recently, also at TriTrypDB (http://tritrypdb.org/; ref. 3), secondly, the systematic analysis of RNA

binding proteins harboring an RNA recognition motif (RRM; ref. 17), and thirdly, tandem affinity

purification of splicing complexes combined with mass spectrometric identification of co-purified

proteins (1, 64). A current list of identified splicing factors is presented in Table 1.

In an excellent previous review on trypanosomatid RNA splicing, Liang et al. described the

discoveries and functional characterizations of the trypanosome spliceosomal U snRNAs and early

characterizations of the corresponding snRNPs (40). This review omits a general discussion of the U

snRNAs and instead focuses on proteins involved in splicing.

U snRNPs and the spliceosome in higher eukaryotes

Our mechanistic insight of RNA splicing and our biochemical and structural knowledge of snRNPs,

splicing factors and the spliceosome stem predominantly from work in the human and yeast systems.


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Unfortunately, there is no uniform nomenclature for the protein factors in the two systems and typically,

there are two distinct names for orthologous factors (listed in ref. 32). In this review, the default is the

denotation from the human system.

The main building blocks of the spliceosome are the U snRNPs whose biogenesis requires several

distinct assembly steps. First, all spliceosomal U snRNAs, except U6, are exported to the cytoplasm

where they bind a set of seven common proteins, known as the Sm proteins B, D1, D2, D3, E, F, and G.

These proteins form a heteromeric ring around a conserved Sm binding site that resides in a single-

stranded region in the 3' terminal domain of the U snRNA. This RNA-protein interaction is typically very

stable and thus, the U snRNA/Sm complex is referred to as the core snRNP. Core snRNP assembly takes

place in the cytoplasm and is linked to U snRNA cap hypermethylation which in turn co-determines the

re-import of the core snRNP into the nucleus. The U6 snRNA does not have a cytoplasmic phase and, in

the nucleus, binds a different complex of seven Sm-like (LSm) proteins termed LSm2-8. Back in the

nucleus, the core snRNPs bind various snRNP-specific proteins, and overall there are approximately 45

different proteins in the human system directly interacting with the spliceosomal U snRNAs (92). As

follows, most of the spliceosome-associated proteins are considered to be non-snRNP proteins.

The RNA sequence determinants for the splicing reaction comprise the 5' and 3' splice sites (SS), and

a branch point (BP) sequence upstream of the 3'SS. In addition, a polypyrimidine tract is typically present

between BP and 3'SS (Fig. 1). Importantly, the spliceosome is assembled step-by-step on the pre-mRNA

and before and during splicing, it undergoes highly dynamic changes in which both RNA and protein

compositions are altered (92 and refs. therein). In brief, the U1 snRNP first recognizes the 5'SS, the

protein factor SF1 the branch point, and the heterodimeric U2 auxiliary factor (U2AF) both branch point

and 3'SS. Subsequently, the U2 snRNP is recruited and the U2 snRNA base-pairs with the BP sequence

displacing SF1, a process which is mediated by the U2-associated protein complexes SF3a and SF3b. At


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this stage, the factor assembly is called pre-spliceosome or complex A. Subsequently, the U4/U6 snRNP

and the U5 snRNP enter the spliceosome in the form of the U4/U6.U5 tri-snRNP which results in the pre-

catalytic complex B. Although in this complex all snRNPs are on board, the spliceosome undergoes major

rearrangements for activation (complex B') including the discard of U1 and U4 snRNPs. After the first

transesterification, the spliceosome is transformed into complex C, and following the second splicing step

it is disassembled. The snRNPs are then recycled for new rounds of splicing. The different spliceosomal

complexes have been purified and biochemically characterized in the yeast and human systems. Besides

the described snRNP changes, these complexes are associated with distinct sets of proteins (Fig. 2;

reviewed in refs. 32, 92).

Is the spliceosome different for SL trans splicing? Interestingly, as first shown for the nematode

Caenorhabditis elegans, the SL RNA splicing substrate itself is assembled into a core snRNP binding the

Sm proteins (12). This finding led to the hypothesis that the SL RNP activates its own splice site and

trans splicing does not require the U1 snRNP (82, 88). Indeed, in vitro studies in the parasitic nematode

Ascaris lumbricoides showed that the destruction of U1 snRNA affected only cis but not trans splicing

(28). Moreover, in the same system, two specific SL RNP proteins were identified and termed SL175 and

SL30 according to their molecular masses (18). Protein-protein interaction experiments suggested that

these proteins bridge the SL RNP, and thus the 5'SS of the SL RNA, via SF1 to the BP, a function which

in cis splicing is mediated by the U1-specific FBP11/Prp40p (human/yeast nomenclature) subunit (18).

SL175 and SL30 are indispensable for SL trans splicing but they have no function in cis splicing or in an

SL-independent mode of trans splicing (18) which has also been described in the human system

(reviewed in ref. 23). While these factors and their interactions are potential anti-parasitic targets, the

amino acid sequences of these proteins are not conserved, and putative orthologs have not been identified

outside of nematodes.


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Trypanosome Sm and LSm proteins, and Sm core variation in U2 and U4 snRNPs

Sm and LSm proteins are small proteins with a molecular mass typically of ~10-20 kDa that share a

highly conserved bipartite Sm motif. The corresponding Sm fold characteristically consists of an N-

terminal helix and a strongly bent, anti-parallel beta-sheet of five strands. While antibodies directed

against the Sm domain of human proteins recognize Sm proteins in a wide range of organisms, they did

not cross-react with trypanosome proteins (56, 62, 63). Hence, it required affinity purification of U

snRNPs and protein analysis to show that trypanosome U snRNAs and the SL RNA bind a set of common

proteins (63). The identity of five of these proteins was revealed in the classic way: U snRNPs were

affinity-purified, amino acid sequence information from common proteins was obtained by protein micro-

sequencing, and the respective genes were cloned with the help of degenerate primers and PCR (66). In

the same study, the missing SmB and SmD3 orthologs, however, could already be identified by mining

the growing T. brucei genome data base (66). Later, this was the exclusive route to identify the orthologs

of LSm2-8 (42). However, while the Sm motifs were readily identifiable in all these proteins, the

remaining amino acid sequences exhibited limited similarity to their putative orthologs in other

eukaryotes and therefore needed functional verification. In case of the Sm proteins, SmG was shown to

complement an SmG-deficient yeast strain (66) whereas the others exhibited protein-protein interactions

which were consistent with the known arrangement in the ring structure (64, 66). For the LSm proteins,

only LSm8 and LSm3 were functionally analyzed at first, and all others identified by sequence similarity

alone (42). This approach backfired because LSm2 and LSm5 ended up to be very interesting Sm proteins

(see below) but not LSm proteins. Eventually, a second study clarified the trypanosome LSm repertoire

and identified new LSm2 and LSm5 orthologs, and provided strong evidence through functional studies

that the correct set of LSm proteins was identified (83). Formation of core snRNPs stabilizes the U

snRNAs and expression silencing of a single Sm or LSm protein leads to a loss of the cognate U snRNA.


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Accordingly, conditional RNA interference experiments targeting each of the seven LSm proteins resulted

in a specific loss of U6 snRNA confirming the new identifications (1, 42, 83).

In initial studies of trypanosome snRNPs, it was found that the trypanosome U2 core snRNP, in

contrast to its human counterpart and other trypanosome U snRNPs, completely disassembled in a cesium

chloride density gradient (15) and in high salt buffers (26). While this instability to salt was originally

attributed to a deviating U2 Sm binding site, which in T. brucei contains an unusual central guanosine

residue, it was found only recently that the different core includes the Sm complex as well. U2 snRNP

purification revealed two U2-specific proteins with apparent sizes of 15 and 16.5 kDa that contained the

bipartite Sm motif (93). This was odd because the whole Sm repertoire had already been characterized

and, moreover, Sm15K, at that time, had been identified as LSm5. However, a comprehensive tagging

and co-immunoprecipitation analysis clarified the issue and showed that Sm15K and Sm16.5k are

paralogs of SmB and SmD3, respectively; they replace these proteins specifically in the U2 Sm core and

do not bind other U snRNAs. Furthermore, snRNP reconstitution assays with recombinant Sm proteins

and synthetic RNAs demonstrated that the guanosine residue of the U2 Sm binding site is the recognition

determinant of the U2-specific Sm core complex (93). In an independent study, the identification of the

two U2-specific Sm paralogs was confirmed and the previously mis-annotated LSm2 was shown to be a

second SmD3 paralog that replaces SmD3 in the U4 snRNP (84). Importantly, the study by Tkacz et al.

(84) provided an in vivo analysis demonstrating that RNAi-mediated expression silencing of the specific

Sm paralogs reduced the abundance of only the corresponding U snRNA. The U4-specific Sm core was

subsequently characterized at the biochemical level verifying the U4-association of the SmD3 paralog

(31). Unfortunately, the studies on Sm core variation established different nomenclatures and Sm15K is

also referred to as specific spliceosomal Sm2-1 protein (SSm2-1), Sm16.5K as SSm2-2, and the U4-

specific protein as SSm4.


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What is the significance of Sm core variation? It has been speculated that it assists U2 and U4 snRNP

assembly (84, 93) and indeed, this has recently been demonstrated for the U2 snRNP. Earlier it was found

that stable protein binding to the 3' terminal region of U2 snRNA, which included the Sm binding site,

was dependent on residues in the 3' terminal loop IV sequence. This suggested that the U2 core snRNP

was not formed by Sm protein binding alone but required cooperative binding of Sm and loop IV-binding

proteins (26). This model was recently verified by the demonstration that the U2-specific Sm15k/16.5K

doublet interacts with the U2 snRNP protein U2A' which in turn interacts with the loop IV-binding

protein U2B'' (70). Only this ternary complex efficiently and specifically interacts with the 3'-terminal U2

snRNA region. In the human system, U2A' is separated from the Sm core by stem-loop III. Since this

structure is completely missing in trypanosome U2 snRNA, it is likely that the Sm15K/16.5K - U2A'

interaction occurs through an essential, parasite-specific protein-protein interface that compensates for the

lack of this RNA structure.

Furthermore, it was suggested that Sm core variation may facilitate snRNP function in the splicing

process (93). For example, human and yeast U2 snRNAs share a conserved motif which is

complementary to the BP sequence. Conversely, in trypanosomes there is no conserved BP sequence and

typically no complementarity between BP and U2 snRNA sequences (46). Possibly, the U2-specific Sm

paralogs undergo specific protein-protein interactions positioning the U2 snRNP at the BP in the absence

of sequence complementarity. A third speculation stated that the different Sm cores may be connected to

different U snRNA cap structures (84). In vertebrate and yeast systems, the U6 snRNA carries a γ-

monomethyl phosphate cap whereas U1, U2, U4 and U5 snRNAs obtain co-transcriptionally an m7G cap

which is further methylated to a 2,7,7 trimethylguanosine (m3G) cap after the formation of the core

snRNP. It was shown that the binding of the Sm proteins to the U snRNAs is a pre-requisite for the

recruitment of the enzyme trimethylguanosine synthase 1 (55, 68). In trypanosomes, U1, U2, U4 and U6


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snRNAs have the same caps as their yeast and human orthologs (19, 58, 65) whereas SL RNA has cap 4

(5) and U5 snRNA lacks a cap (21, 96). Theoretically, Sm core variation could facilitate the recruitment

of different cap-modifying enzymes into the core RNP but there is no correlation between the type of cap

and the type of Sm core. For example, U1, U2, and U4 snRNAs bind different Sm complexes but share

the same m3G cap. Moreover, it was demonstrated experimentally for the T. brucei U2 snRNA that cap

trimethylation does not depend on the presence of the Sm binding site nor on formation of the core RNP,

thus excluding the possibility that the U2-specific Sm core is involved in cap hypermethylation (25).

SMN-mediated assembly of canonical Sm cores

When Wang et al. (93) reconstituted core snRNPs with recombinantly expressed Sm proteins, they

detected specific binding of the canonical and U2-specific Sm cores to their cognate Sm binding sites only

with short RNA fragments whereas full-length U snRNAs did not discriminate between the two Sm

complexes. This suggested that other activities in the cell confer specificity of Sm core binding. A

candidate for such an activity was the SMN (survival motor neuron) complex, which in the human system

was shown to act as a catalyst for core snRNP formation (reviewed in refs. 34, 60). The human SMN

complex consists of the SMN protein and seven additional subunits, termed Gemin2-8, and it binds the

SmD1/D2-E/F/G and SmD3/B sub-complexes in an open ring formation (14). This interaction then leads

to U snRNA binding, ring closure, and dissociation of the SMN complex. While standard annotation of

the TriTryp genomes did not identify SMN or Gemin homologs, tandem affinity purification of the T.

brucei SmB protein (see below) led to the identification of highly divergent orthologs of SMN and

Gemin2 (64). No other Gemin orthologs were found and it is possible that they do not exist in

trypanosomes because lower eukaryotes in general have a strongly reduced Gemin repertoire and, in

Drosophila melanogaster, the SMN/Gemin2 complex was sufficient to mediate core RNP assembly in

vitro (37). In trypanosomes, in vitro core snRNP assembly experiments functioned efficiently in the


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absence of SMN but when the factor was added it exhibited a striking discriminatory role: in its presence

the canonical Sm core was efficiently loaded onto its cognate U5 snRNA but not onto U4 and U2

snRNAs nor onto a U5 snRNA with a mutated Sm site (64). In contrast, the SMN complex had no effect

on the binding of the U2-specific Sm core. These findings suggested that the SMN complex specifically

bound the canonical SmD3/B subcomplex and directly interacted with SmB because this protein, in

contrast to SmD3, is replaced in both the U2 and U4-specific Sm cores. This was indeed the case. SMN

purification co-isolated only the SmB and SmD3 proteins and not their paralogs, and pull-down assays

with recombinant, tagged SMN proteins identified a direct interaction with SmB and the N-terminal part

of SMN (64). The latter finding, again, is highly significant because it identified an important, potentially

parasite-specific protein-protein interaction: human SMN utilizes an internal Tudor domain and C-

terminal regions to interact with dimethylated arginines in the RG-rich C-termini of Sm proteins (74)

whereas in trypanosomes neither Tudor domain nor RG-rich domains are present in SMN and Sm

proteins, respectively (64). Another striking difference to the human system was found in regard to SMN

localization. In the human system, core snRNP assembly takes place in the cytoplasm and, accordingly,

human SMN is primarily localized in this compartment. Conversely, trypanosome SMN was found almost

exclusively in the nucleus suggesting that U snRNP assembly in this organism is nuclear, a finding which

is consistent with localizations of SL RNA and U2 snRNA by fluorescence in situ hybridization (9, 84).

In summary, it appears that despite its small size, the trypanosome SMN complex is mechanistically

complex entailing both chaperone and specificity functions in core snRNP assembly.

If the SMN complex only chaperones the assembly of the canonical Sm core, how are then the U2 and

U4-specific Sm cores put together? One possibility is that they require a different, yet to be determined

assembly chaperone. Alternatively, the specific interactions of the U2 Sm paralogs SmK15/SmK16.5 with

U2A'/B'' may facilitate correct assembly of the U2-specific Sm complex onto the U2 Sm binding site. On

the other hand, U4 core snRNP formation appears to be independent of snRNP-specific proteins because


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in core RNP reconstitution assays, SSm4 alone determined efficient and specific assembly of the U4-

specific Sm complex onto the U4 snRNA (31).

Tandem affinity purification of splicing complexes in T. brucei

Until recently, only two snRNP-specific proteins had been studied in trypanosomes, namely the

orthologs of human U2A' (originally termed U2-40K; ref. 16) and the U5-specific PRP8 (45). While the

latter was identified by sequence homology, U2A' was co-purified with the U2 snRNA at high stringency

U snRNP purifications which typically left only the core structures intact (63). For a more comprehensive

biochemical characterization of U snRNPs and/or of the spliceosome it was therefore essential to purify

the RNA-protein complexes at conditions of lower stringency. A method well-suited for this purpose is

tandem affinity purification (TAP) which is based on expressing a known protein factor fused to a

composite TAP tag. TAP comprises two consecutive high affinity chromatography steps which are carried

out at nearly physiological conditions. Since the advent of this technology (72), the TAP tag and the TAP

method have been modified in various ways to accommodate different systems, extracts or protein

complexes (27). For the purification of nuclear protein complexes in trypanosomes, the PTP modification

of TAP has proven to be very useful (27, 73). One of the first applications of the PTP tag was the

purification of the trypanosome U1 snRNP. A first characterization of this snRNP had revealed a protein

with sequence homology to the human U1-70K protein (65). And indeed, PTP-tagging and purification of

T. brucei U1-70K-PTP resulted in the specific co-purification of the U1 snRNA (67). The protein profile

of the purification comprised the Sm proteins, the tagged protein, two additional proteins of major

abundance and several proteins of minor abundance. The proteins of the two major bands were identified

by mass spectrometry and found to be annotated as conserved hypotheticals meaning that they were

conserved among trypanosomatids but had no obvious similarity to proteins of other eukaryotes.

However, when the kinetoplastid sequences of one of the new proteins was compared to those of known


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U1-specific proteins of model organisms, it was identified as the ortholog of human U1C (67). In contrast,

the second protein, termed U1-24K, could not be meaningfully aligned to known U1 proteins and

therefore likely represents a trypanosome-specific U1 snRNP subunit.

Since this initial tandem affinity purification of a snRNP was successful, more comprehensive

proteomic analyses of trypanosomal splicing complexes were carried out by PTP tagging and purification

of the common proteins SmD1 (1) and SmB (64). Overall, mass spectrometry identified 53 proteins in

these purifications and the majority of the proteins co-purified in both studies (Table 1). Moreover, with

the exception of three LSm proteins and two non-snRNP proteins, all known trypanosomal snRNP

proteins were identified in these proteomic analyses confirming the high significance of the proteomic

data sets. Consequently, bioinformatic analyses of the amino acid sequences of un-annotated proteins

revealed several new orthologs of known splicing factors (Table 1) and for LSm2 (U6), U1A, PRP4 (U4),

and U5-40K, the bioinformatic identifications were confirmed by co-immunoprecipitation experiments

which showed that these proteins were bound to their predicted snRNAs (1).

While these proteomic analyses increased the number of spliceosomal protein orthologs in

trypanosomes considerably and identified potentially novel splicing factors (see below), the number of

proteins that co-purified with SmD1 or SmB are ~3 fold lower than the protein count in human

spliceosomes. Proteomics of yeast spliceosomal complexes revealed about 90 proteins (22) which is

lower than the count in the human system but still about twofold higher than the identified trypanosome

repertoire. One possible interpretation of this finding is that the splicing machinery of early-diverged

trypanosomatids is simplified. However, this is unlikely because the vast majority of newly identified

proteins are snRNP proteins, and non-snRNP proteins are strongly underrepresented (Fig. 2). In fact,

trypanosome orthologs have been identified for nearly all known bona fide snRNP proteins indicating that

a trypanosome spliceosome comprises additional non-snRNP proteins possibly in comparable numbers to


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those in yeast and humans. Hence, the question arises why only a few non-snRNP proteins co-purified

with SmB and SmD1. Both proteomics studies were carried out according to the standard PTP protocol

which includes the extract preparation procedure (38, 73). Since these extracts contain an estimated

overall salt concentration of 250-300 mM, it is likely that the spliceosome did not withstand the extract

preparation procedure. Accordingly, a sucrose gradient sedimentation analysis of SmD1-PTP-purified

material showed that the U snRNPs did not co-sediment as part of a larger complex and complexes with S

values greater than 20 were not detected (1). In contrast, spliceosomal 45S complexes were characterized

by a combination of glycerol gradient sedimentation and native gel electrophoresis in extracts of lower

salt concentration (41). It is therefore likely that modifying the extract preparation procedure will result in

formation of higher order spliceosomal complexes which possibly can be isolated by tandem affinity

purification and characterized by mass spectrometry in the future.

As discussed above, in both proteomic studies, the highly divergent SMN and Gemin2 orthologs co-

purified. To better understand the trypanosome SMN complex, Palfi et al. (64) PTP-tagged and tandem

affinity-purified both proteins, and identified co-purified proteins by mass spectrometry. While no other

Gemin proteins were detected, which supports the idea that a SMN/Gemin2 complex is sufficient for

chaperone function, surprisingly, all coatomer subunits co-purified. While the coatomer complex

functions in vesicular transport between Golgi and endoplasmic reticulum (48), the significance of the

coatomer-SMN/Gemin2 interaction is not understood. Possibly, the trypanosome SMN/Gemin2 complex

has a cytoplasmic function independent of snRNP core assembly (64) or there is a cytoplasmic component

of the core snRNP assembly process which is vesicular and has not been detected yet.

Analysis of proteins carrying an RRM

Besides by tandem affinity purification, trypanosome splicing factors have been identified through a

focus on RRM-containing proteins. Since trypanosomatid protein coding genes are typically arranged in


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long tandem gene arrays which are transcribed polycistronically, differential gene expression is typically

regulated post-transcriptionally, for example at the level of RNA stability. Many proteins which affect

RNA stability bind to mRNAs directly by virtue of an RRM motif and thus, RRM-containing proteins

have become a research focus in gene expression studies in both T. brucei and Trypanosoma cruzi (17).

Since the spliceosome comprises several RRM proteins, their identification came as a benefit from the

attempt to determine the role of RRMs in gene expression regulation.

One RRM protein that was identified as a splicing factor was a subunit of the U2-associated SF3b

complex. SF3a and SF3b are two essential multi-subunit splicing factors that interact with the U2 snRNP

after its recognition of the BP. The trypanosome protein was identified as the ortholog of human SF3b49

and accordingly, expression silencing of the corresponding gene was lethal and affected RNA splicing in

T. brucei (51). Moreover, TAP-tagging and purification of the protein, using the original TAP method, led

to the complete characterization of the trypanosome SF3b complex. Orthologs of all seven human SF3b

subunits were identified including the RRM protein SF3b14, often referred to as p14 (51). The SF3a

complex appears to be also present in trypanosomatids because a putative homolog of the SF3a60 subunit

was annotated in the genome database (Table 1).

Other RRM proteins that were found to be splicing factors comprise the snRNP protein U1A (1) and

the U2 auxiliary factor components U2AF65 and U2AF35 (90, 91). In addition, RRM-containing serine-

argine-rich (SR) proteins have been identified. SR proteins comprise a phylogenetically conserved protein

family and, as has been shown in other systems, play significant roles in constitutive and alternative

splicing of pre-mRNA (reviewed in ref. 43). SR proteins contain one or two N-terminal RRMs and a C-

terminal RS domain, rich of arginine-serine dipeptides. The first such protein discovered in trypanosomes

was termed RRM1 (52). While RRM1 was shown to be encoded by an essential gene and located in the

nucleus, its specific function has not been determined yet (53). A second SR protein, termed


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trypanosomal SR-rich protein 1 (TSR1), was localized to the nucleus, was shown to bind to the

heterologous human U2AF complex and, in a yeast three hybrid system, it appeared to interact with the

SL RNA (29). While these findings led to the speculation that TSR1 may facilitate recognition of the SL

RNA by the trans spliceosome (29), a functional characterization of TSR1 strongly indicated that the

factor has an essential role in cis splicing but not in SL trans splicing (Christian Tschudi, Yale University,

personal communication). This result is in accordance with a previous study of the T. cruzi ortholog TcSR

which showed that TcSR was functional in cis splicing in a heterologous system (69). Finally, RRM

protein analysis in trypanosomes revealed two homologs (PTB1 and PTB2) of the mammalian

polypyrimidine tract binding protein. While mammalian PTB did not co-purify with spliceosomal

complexes and has several non-splicing functions, it negatively affects the splicing process presumably by

binding to the polypyrimidine tract near the 3'SS thereby interfering with U2AF65 function (78).

Functional characterization of trypanosome PTB1 and PTB2 did not reveal a repressor function of these

proteins in splicing. In contrast, a detailed study provided very strong evidence that both proteins are

essential for trans splicing of pre-mRNAs that contain C-rich polypyrimidine tracts (79). In addition,

expression silencing of PTB1, but not of PTB2, affected cis splicing indicating that both proteins have

distinct activating functions in trypanosome RNA splicing (79).

Bioinformatic identification of trypanosome splicing factors

A third route to identify RNA splicing factors has been data mining. Some of the splicing factors in

trypanosomes are conserved enough to be identified by in silico analysis alone. For example, it was

straightforward to identify the missing orthologs of the human snRNP proteins Snu13 and U5-200K for

this study (Table 1). Similarly, the important CDC5 subunit of the PRP19 complex, which is an essential

component of the active spliceosome, was readily identifiable in the trypanosome genome database (Table

1). Two splicing factors which had previously been identified bioinformatically are PRP43 and PRP31


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(41). PRP43 is a conserved spliceosomal helicase with essential functions in intron lariat release from the

spliceosome (54) and in spliceosome disassembly (2) whereas PRP31 is a factor of the U4/U6.U5 tri-

snRNP being important for tri-snRNP formation and assembly into the spliceosome (50). The

trypanosome PRP31 and PRP43 appear to be functionally equivalent because both proteins were shown to

be essential for both cis and SL trans splicing, and PRP31 was specifically associated with the

trypanosome U4/U6.U5 tri-snRNP (41). Nevertheless, the bioinformatics route of identifying

trypanosome splicing proteins has not been exploited extensively and it is very likely that a systematic

approach will reveal additional [putative] orthologs of non-snRNP proteins.

Overall, our knowledge of the spliceosomal protein repertoire of trypanosomes has greatly increased

in the past years. While the set of snRNP proteins appears to be nearly complete, most of the non-snRNP

factors have probably not been identified yet. However, the identification of individual components of

spliceosomal protein complexes such as PRP19 and SF3a (Fig. 2) indicate that these complexes are

present and that they can be further analyzed. For example, in yeast more than twenty splicing proteins

were identified by tandem affinity purification of the CDC5 ortholog Cef1p (reviewed in ref. 32). The

identification of CDC5 in this study will enable a comparable analysis in trypanosomes.

Another important aspect of the newly identified trypanosome splicing factors is that they strongly

indicate that trypanosomes form a spliceosome that possesses the same basic components and undergoes

the same dynamic rearrangements as its human and yeast counterparts. It should be kept in mind that with

the exception of a 45S spliceosome detection by native gel electrophoresis (41) there is so far no

biochemical evidence yet that trypanosomes do form complexes that correspond to the well-characterized

spliceosome E, A, B, or C complexes in the yeast and human systems. On the other hand, a comparison of

human proteins that enter the spliceosome at these defined stages and of the known trypanosome


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repertoire shows that for each spliceosome transition characteristic trypanosome orthologs have been

identified (Fig. 2).

Trypanosome-specific aspects of the spliceosome

Despite the rapidly increasing number of identified trypanosomal RNA splicing factors, only a few

functional protein characterizations have been published thus far. Nevertheless, several trypanosome-

specific characteristics of the splicing machinery have already been identified. As discussed above, Sm

core variation, including the trypanosome-specific interaction between SmB and SMN, as well as the

particular architecture of the U2 RNP core, involving potentially unique interactions between

Sm15K/16.5K and U2A', are trypanosome-specific U snRNP features. Other notable differences, shown

in the T. cruzi system, include the demonstration that the U2AF subunits U2AF35 and U2AF65 exhibit

weak or no interaction (91), that instead U2AF65 forms a stable complex with the BP binding protein SF1

(91), and that, within the U2-related SF3b complex, the protein interface between the SF3b155 and

SF3b14 subunits appears to be larger and more complex than in the human system (4).

Another interesting trypanosome splicing factor is U5-Cwc21. This protein shares a highly conserved

N-terminus with the human SRm300/SRRM2 protein and yeast Cwc21p (complexed with Cef1p protein

21). Co-immunoprecipitation analysis showed that the trypanosome protein is predominantly associated

with U5 snRNA, and expression silencing of U5-Cwc21 was lethal and affected both cis and trans

splicing (1). In contrast, yeast Cwc21p and human SRm300 have redundant, non-essential roles in RNA

splicing because CWC21 is a non-essential gene and SRm300 can be immunodepleted from extract

without affecting splicing efficiency in vitro (10). Moreover, while yeast Cwc21p does interact with the

U5-protein PRP8 (24), it is predominantly associated with U2 snRNA and not with U5 snRNA (33).


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These findings therefore strongly indicate that trypanosome U5-Cwc21 has an essential function in RNA

splicing that is unique to trypanosomes.

A further peculiarity of trypanosome splicing factors is the expression level of U1 snRNP

components. In the nematode system, the U1 snRNP exclusively functions in cis splicing and not in SL

trans splicing (28). If the trypanosome U1 snRNP functions analogously, it would be required only for the

removal of a single intron from two different pre-mRNAs. However, the U1-specific proteins U1-70K,

U1-24K, and U1C are among the most abundant proteins that co-purified with SmD1 (1). This

discrepancy between intron number and U1 snRNP expression level suggests that the trypanosome U1

snRNP has functions beyond intron removal. There is evidence that the trypanosome 45S spliceosome

contains both SL and U1 snRNA and it was suggested that there may only be one kind of spliceosome for

both cis and trans splicing (41). If this is true, the U1 snRNP may be essential for spliceosome integrity or

it may have a yet undetected, trypanosome-specific function in trans splicing. Alternatively, the

trypanosome U1 snRNP, as its human counterpart, may function beyond intron removal in transcription

initiation and/or elongation (reviewed in ref. 6).

Finally, there seems to be a difference of SL RNP recruitment to the BP between nematode and

trypanosome systems. While the nematode SL RNP apparently docks on SF1 via a protein bridge (18),

immunoprecipitation of trypanosome SF1 at low stringency conditions did not co-precipitate SL RNA

(D.L. Ambrósio & A. Günzl, unpublished results). It is therefore likely that other proteins and protein-

protein interactions than in the nematode system mediate the recruitment of the trypanosome SL RNP.

Possibly, U5 and U6 snRNPs play a role in this process because U5 and U6 snRNAs were convincingly

shown to interact with the 5'SS of the SL RNA (94, 97). If SL RNP recruitment to pre-mRNA requires

trans splicing-specific factors as in the nematode system, potential candidates of such proteins are listed

in Table 1; there are currently eight proteins which co-purified with trypanosomal splicing complexes but


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could not be annotated convincingly. Three of these proteins may be the orthologs of cyclophylins and of

the PRP19-related factor Cwc15 although the sequence similarities are very weak. The remaining five

proteins are novel in sequence because they do not exhibit any sequence similarity to non-trypanosomatid

proteins.

Perspectives

The spliceosome is one of the most complex molecular machineries in the cell and it is a great

challenge to functionally characterize this dynamic RNP-protein machinery. In the past years, major

progress has been made in the biochemical and structural analysis of the human spliceosome (47, 92). If

corresponding studies can be carried out in trypanosomes, it will be possible to determine in detail

essential differences between trypanosome and human spliceosomes. While such differences may be the

consequence of evolutionary divergence or may represent SL trans splicing-specific requirements, they

are potential anti-parasitic drug targets. This notion is not remote since the spliceosome has been validated

as a drug target, for example for anticancer treatment (reviewed in ref. 86). Although the undertaking of

comprehensively analyzing the trypanosome spliceosome appears overwhelming, the prospects are

nevertheless good because all necessary tools are in place. As shown in figure 2, there are now several

new spliceosomal proteins which can serve as baits in tandem affinity purification to broadly characterize

the trypanosome splicing factor repertoire. The conditional RNAi-based expression silencing system in T.

brucei (95) in combination with established RT-PCR and primer extension assays for the analysis of trans

and cis splicing defects provides an in vivo platform for determining splicing functions of individual

proteins. Moreover, a homologous in vitro trans splicing system was recently established in T. brucei

which will allow the functional dissection of important splicing factors (75). Finally, the recent

demonstration that the tandem affinity-purified trypanosome transcription factor complex TFIIH was


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sufficiently intact and pure to determine its molecular structure by macromolecular electron microscopy

(39) strongly indicates that similar structures can be obtained from tandem affinity-purified splicing

complexes.

And there is another potentially exciting perspective. While introns and alternative splicing greatly

enhance the protein repertoire in higher eukaryotes (recently reviewed in ref. 61), the functional role of

introns in lower eukaryotes is not well understood. Trypanosomes appear to have reduced their intron

repertoire to only two (76). Why did they not eliminate these two introns as well? The fact that the

insertion site of the PAP intron is conserved in trypanosomatids argues that the intron has a specific and

essential function which was retained throughout trypanosomatid evolution. Since it should be

straightforward to test the outcome of [conditionally] deleting these intron sequences in the trypanosome

genome, it may be possible to determine the specific function of these introns and understand the

functional significance of cis splicing in these early-diverged organisms.

ACKNOWLEGMENTS

I am thankful to Christian Tschudi (Yale University) for communicating unpublished data and to Tu

N. Nguyen and Daniela L. Ambrósio for critical reading of the manuscript. This work was supported by

National Institutes of Health R01 grants AI059377 and AI073300 to A.G.


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Figure Legends

FIG. 1. Schematic of the mammalian cis splicing and the trypanosome SL trans splicing reactions.

Upstream exon and spliced leader are drawn as grey rectangles, and downstream exon and the

trypanosome gene are drawn as black rectangles. 5' and 3' splice sites (SSs) are represented by small open

boxes, branch points (BPs) by closed circles, polypyrimidine tracts by small striped boxes, and the cap 4

structure of the spliced leader as an oval. Conserved sequences are provided below the drawing with

invariant residues underlined. While in mammalian systems, 5'SSs, BPs and 3'SSs exhibit partly

conserved sequences (R, purine; Y, pyrimidine, N, any base), there is no obvious sequence conservation

at trypanosome BPs (44) and 3'SSs, although it was shown for the latter that an AC dinucleotide (*)

preceding the AG residues drastically reduces splicing efficiency unless a compensatory AG dinucleotide

is present within the 5' untranslated region (77). It appears that the importance of the polypyrimidine tract

becomes more important when consensus sequences are lacking. Yeast has highly conserved splice site

and BP sequences and some yeast introns function without a polypyrimidine tract (not shown). The partly

conserved sequences in mammals require a small polypyrimidine tract in the range of 10 to 12 residues

(Y10-12) whereas in trypanosomes the polypyrimidine tract is large, an essential sequence determinant for

efficient splicing, and it starts typically right downstream of the BP (44, 77). After the first

transesterification reaction, cis splicing results in a lariat intron structure whereas a Y-structure

intermediate is formed in the SL trans splicing process. After the second transesterification, these intronic

structures are debranched (not shown) and rapidly degraded.

FIG. 2. Comparison of known spliceosomal factors of humans and trypanosomes. Schematic drawing of

spliceosomal complexes during a splicing reaction as described in the mammalian and yeast systems. For

each complex, proteins are listed that enter the spliceosome at the outlined stage (slightly modified human

protein repertoire according to ref. (92). Please note that only incoming proteins are listed and proteins


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leaving the spliceosome in the transitions are not recognized. Bold, blue lettering indicates proteins for

which orthologs have been found in trypanosomes whereas red lettering specifies trypanosome-specific

factors. 1, Highly divergent, putative cyclophilin orthologs have been co-purified with SmD1 and SmB1

(Table 1). 2, U5-100K is a DExD/H-box helicase and it is unclear whether one of the putative

trypanosome DExD/H-box helicases (Table 1) represents a U5-100K ortholog. 3, U5-Cwc21 is possibly

the ortholog of human SRM300 but seems to have a trypanosome-specific function (see text). 4, The

trypanosome exon junction complex has recently been characterized (7) but its specific function in RNA

splicing or metabolism remains unclear.


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Table 1: Spliceosomal proteins of Trypanosoma brucei

ANNOTATION 1 ACCESSION NO 2 Mr (kDa) TAP 3 REF./E VALUE 4 ANNOTATION 1 ACCESSION NO 2 Mr (kDa) TAP 3 REF./E VALUE 4

Sm/LSm proteins PRP19 complex SmB Tb927.2.4540 12.3 1,2,3,4 (66) PRP19 Tb927.2.5240 54.3 1,3,4 E = 3e-42 SmD1 Tb927.7.3120 11.7 1,2 (66) * CDC5 Tb927.5.2060 80.1 - E = 1e-30 SmD2 Tb927.2.5850 12.5 1,2 (66) CRN Tb927.10.9660 87.7 1 E = 7e-41 SmD3 Tb927.4.890 12.4 1,2,3,4 (66) SYF1 Tb927.5.1340 92.2 1 E = 7e-23 SmE Tb927.6.2700 9.6 1,2 (66) ISY1 Tb927.8.1930 31.7 1 ISY1 domain E = 6e-7 SmF Tb09.211.1695 8.4 1,2 (66) KIAA1604/Cwc22 Tb11.01.2520 66.82 2 E = 2e-49. SmG Tb11.01.5915 8.9 1,2 (66)

SSm2-1/Sm15K Tb927.6.4340 12.8 1,2 (84, 93) un-annotated proteins that co-purified in spliceosomal complexes 5 SSm2-2/Sm16.5K Tb927.10.4950 14.7 1,2 (84,93) cons. hypo. Tb927.8.6280 27.1 1,2 putative cyclophilin (64) SSm4 .Tb927.7.6380 23.2 1,2 (84) cons. hypo. Tb927.8.2090 21.6 2 putative cyclophilin (64)

LSm2 Tb927.8.5180 13.2 1,2 (1, 83) cons. hypo. Tb927.10.11950 22.4 2 putative Cwc15 (64) LSm3 Tb927.7.7380 10.1 - (42) cons. hypo. Tb927.8.4790 26.0 1 novel LSm4 Tb11.01.5535 14.2 1,2 (42) cons. hypo. Tb927.2.3400 42.0 1 novel LSm5 not assigned 6 12.0 - (83) cons. hypo. Tb927.7.1890 31.0 1 novel LSm6 Tb09.160.2150 9.1 - (42) cons. hypo. Tb927.5.2910 20.0 1 novel LSm7 Tb927.5.4030 10.2 1,2 (42) cons. hypo. Tb11.02.0465 12.1 1 novel LSm8 Tb927.3.1780 14.0 1,2 (42) annotated proteins w/o known splicing function that co-purified in spliceosomal complexes

SMN/Gemin2 and associated proteins eEF-1α Tb927.10.2100 49.1 2 SMN Tb11.01.6640 17.0 1,2,3,4 (64) HSP70 Tb11.01.3110 75.4 1,2,3 Gemin2 Tb927.10.5640 55.4 1,2,3,4 (64) Importin α Tb927.6.2640 58.0 2 Coatomer α Tb927.4.450 132.0 3,4 (48) La protein Tb927.10.2370 37.7 1,2,3 Coatomer β Tb927.1.2570 110.0 3,4 (48) NORF1 Tb927.5.2140 93.3 1 Coatomer β' Tb927.2.6050 93.9 3,4 (48) PABP1 Tb09.211.2150 62.1 1,2 Coatomer γ Tb11.01.3740 97.5 3 (48) TRYP1 Tb09.160.4250/80 22.4 1 Coatomer δ Tb927.8.5250 57.3 3,4 (48) Coatomer ε Tb11.01.6530 34.8 3,4 (48) miscellaneous splicing factors Coatomer ζ Tb927.10.4270 20.5 3 (48) U2AF35 Tb927.10.3200 29.1 - (90) U2AF65 Tb927.10.3500 96.6 - (91)

U1 proteins SF1 Tb927.10.9400 31.6 - (91) U1-70K Tb927.8.4830 31.7 1,2 (67) PRP17 Tb927.3.1930 52.8 2 E = 4e-59 U1A Tb927.10.8280/8300 18.0 1 (1) PRP31 Tb927.10.10700 39.7 - (41)

U1-24K Tb927.3.1090 24.2 1,2 (67) PRP43 Tb927.5.1150 82.9 - (41) U1C Tb927.10.2120 21.7 1,2 (67) PTB1 Tb09.211.0560 37.0 - (79) PTB2 Tb11.01.5690 54.7 - (79)

U2 proteins TSR1 Tb927.8.900 37.5 - SR-like protein (29) U2A' (U2-40K) Tb927.10.2120 36.5 1,2 (16) RRM1 Tb927.2.4710 50.0 - SR-like protein (52) U2B'' Tb927.3.3480 13.6 1,2 (70) SR protein Tb09.160.5020 17.6 - TriTrypDB, E = 3e-06 SF3a60 Tb927.6.3160 61.5 - TritrypDB, E = 1e-13 SF3b(SAP)155 Tb11.01.3690 122.0 - (4, 51) putative spliceosomal DExD/H-box helicases SF3b(SAP)145 Tb927.6.2000 52.5 - (51) Tb927.6.4600; Tb927.6.4600; Tb927.10.5280; Tb927.10.7280; Tb927.10.9130; Tb11.02.3460; SF3b(SAP)130 Tb927.7.6980 195.0 1 (51) Tb927.7.7300; Tb11.02.1930 SF3b(SAP)49 Tb927.3.5280 29.8 - (51) SF3b(SAP)14b Tb927.10.7390 12.7 - E = 6e-12 (51) putative spliceosomal peptidyl-prolyl cis/trans isomerases SF3b(SAP)10 Tb09.211.2205 10.4 - SF3b10 domain, E= 4e-7 (51) >20 candidates SF3b14 (p14) Tb927.10.7470 13.3 - (4, 51) U4 proteins 1 Annotation according to the human system. PRP3 Tb09.160.2900 63.2 1,2 PRP3 domain, E = 2e-42 2 Accession numbers of the TriTrypDB data base (http://www.tritryp.org/). PRP4 Tb927.10.960 65.5 1,2 (1) 3 Protein was co-tandem affinity-purified with SmD1 (1), SmB (2), SMN (3), or Gemin2 (4).

* Snu13 Tb09.160.3670 13.6 - E = 5e-34 4 Protein sequences of identifications without experimental support were compared to the human genome and E values determined by NCBI BLAST. REF., references.

U5 proteins 5 E-values lower than 1e-05 were considered to be not significant. PRP8 Tb09.211.2420 277.0 1,2,4 (45) 6 The gene of LSm5 has not been recognized as a protein coding gene yet.

* U5-200K Tb927.5.2290 249.3 1 E ≈ 0

U5-102K Tb11.01.7330 111.0 1 E = 1e-5 Mr, molecular mass. U5-116K Tb11.01.7080 105.5 1,2 E = 8e-100 Asterisks mark genes which have been annotated in this study. U5-40K Tb11.01.2940 35.0 1,2 (1) Trypanosome-specific proteins are shaded in gray. U5-15K Tb927.8.2560 17.7 1 E = 4e-26

U5-Cwc21 Tb09.160.2110 16.2 1,2 (1)


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