Post on 19-Dec-2015
transcript
If RNA can be bothcatalyst and genome,
RNA could replicate itself… the first “living” molecule!
catalytic(+) strand
template(–) strand
catalytic (+) strand copies unfolded template (–) strand
two catalytic (+) strandsand original template (–) strand
Note that addition of a pppN to the 3' OH of a growing
RNA chain is nearly identical, chemically, to attack of a 3'
OH on the 5' or 3' splice site!
How the RNP got its protein…an offer it could not refuse?
independent RNAprotein makes an offer the RNA cannot refuse
RNA relaxes, now protein-dependent
Bruce Alberts (1986) The function of the hereditary materials: Biological catalyses reflect the cell's evolutionary history. Am Zoologist 26, 781-796
ribosome
spliceosomereverse transcriptase
metabolism
Catalytic RNA could have replicated itself before the advent of protein
synthesis!
Invention of the ribosome createda ribonucleoprotein (RNP) worldcomposed of RNA + proteins.
Invention of the spliceosome createdcomplicated proteins by exon shuffling.
Invention of reverse transcriptase began the transition from RNA genomes to DNA genomes, with retroviral-like elements as transitional genomes that persist today.
The RNP World is alive and well in our cells, but we are distracted by the vast genetic storage capacity of DNA!
excess iron sequestered by ferritin
scarce iron imported by transferrin receptor
Rouault and Klausner (1996) TiBS 21, 174; Beutler (2004) Science 306, 2051; Du et al. (2008) Science 320, 1088.
Translational control is powerful,and many proteins have "second jobs"
5’ UTR
3’ UTR
RNA is more than a structure or informational sequence…key steps in the "Central Dogma" are catalyzed and regulated by RNAs
DNA mRNA protein
mRNA splicingcatalyzed by small nuclear RNAs(snRNAs)
translation of mRNAs catalyzed byribosomal RNAs(rRNAs) and tRNAs
mRNA transcriptionregulated by long noncoding RNAs
(lncRNAs)chromatin structure regulated by small interfering RNAs(siRNAs and piRNAs)
defensive RNAs biosynthetic and regulatory RNAs
mRNA translation regulated
by microRNAs (miRNAs)and competing endogenousRNAs (ceRNAs)
The amazing world of RNA interference (RNAi)
miRNAs, siRNAs, ceRNAsand their relatives
Ghildiyal and Zamore (2009) Small silencing RNAs:an expanding universe. Nat Rev Genet 10, 94-108; Broderick and Zamore (2011) MicroRNA therapeutics. Gene Therapy 18, 1104-1110.
RNA interference (RNAi)
Fighting fire with fireor
How Nature uses RNAs
to regulate themselves,
chromatin structure, and transcription
2006 Nobel Laureates Andrew Fire (center), Craig Mello (right),
and Alfred Nobel (left, unsmiling)
Caenorhabditis elegans(aka C. elegans, "the worm", nematode, roundworm)
1 mm
Zamore and Haley (2005) Ribo-gnome: The big world of small RNAs. Science 309, 1519-1524; Ghildiyal and Zamore (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet 10, 94-108
The human genome is chock full of two dispersed, highly repeated families of retroelements called L1's or "LINEs" (long interspersed repeated elements) and Alu's or "SINEs" (short interspersed repeated elements). L1 and Alu elements both contain promoters, and RNA transcripts of these elements are reverse transcribed into DNA copies which insert into new, almost random sites throughout our genome. L1 elements encode a reverse transcriptase and DNA integrase that enables them to move to new sites, whereas Alu elements — which have no protein coding capacity — must borrow the L1 reverse transcriptase and integrase in order to retrotranspose.
The big question: Why aren't our genomes overun or destroyed by this infestation of mobile, parasitic DNA elements? Use RNA to fight RNA!
Alu RNA
Alu RNA
genomic DNA Alu geneL1 L1
L1 RNA
L1 RNA
Alu Alu
Alu RNA
The human genome is chock full of parasitic retrotransposable elements (“retrotransposons” aka “retroposons”) that use a
reverse transcriptase to replicate and transpose through RNA intermediates
LINEs
SINEs
autonomous
non-autonomous
autonomous
non-autonomous
850,000
1,500,000
21%
13%
endogenousretrovirus-like
elements450,000 8%
6-8 kb
}6-11 kb
1.5-3 kb
100-300 bp
AAAORF1 RT-INT
AAA
gag RT-INT env
gag
DNA transposonfossils
autonomous
non-autonomous300,000 3%
2-3 kb
80-3,000 bp}transposase
( )
length
copy numb
er
percentage
of genome
structure of retroelemen
tclass
autonomy
integration
retroviral particle
extracellular
intracellular
RNA
RNA
retroviral particle
RNA
cDNA
DNA
cDNA
RNA
reverse transcriptio
n
second strand synthesis
transcription
provirusgenome
translation to generate reverse transcriptase (RT), integrase, and capsid proteins for viral
assembly
integration
RNA
RNA
cDNA
DNA
cDNA
RNA
reverse transcriptio
n
second strand synthesis
transcription
parental retroelementgenome
translation of LINE RNA to generate reverse transcriptase
and integrase also used by noncoding SINEs
daughter retroelement
+RT
RNA
+RT
LINEs and SINEs may move from cell to cell, organism to organism,and species to species,as stowaways in viral
particles.
stowaway RNAs
Retroviruses and retrotransposable elements made (ridiculously) simple
Retroposition of SINEs and LINEs requires portable promoter and termination signals within the RNA transcription unit: this is possible because the transcription initiation factors that position RNA polymerase at the initiation site can do their job from upstream or downstream, and the polyadenylation signal is transcribed and preserved within the mature RNA transcript.
RNAPTIF
+1
RNAP TIF
+1
“conventional” externalpromoter
internal“portable”promoter
pA
pA
AAAAA
AAAAA
Surprisingly few diseases are caused by spontaneous retroposition of SINEs and LINEs
Although 1,500,000 SINE insertions and 850,000 LINE insertions account for >34% of the complete human genome sequence, SINEs are responsible for only 25 or 0.05% of all known disease-causing mutations, and LINEs for only 5 or 0.01% of the total.
Callinan and Batzer (2006) Retrotransposable elements and human disease. Genome Dyn 1, 104; Plasterk (2002) RNA Silencing: The Genome's Immune System. Science 296, 1263
Most of these 1,500,000 SINE insertions and 850,000 LINE insertions have degenerated in place by neutral mutation and are no longer mobile. The few mobile SINEs and LINEs that have caused spontaneous disease are “young,” i.e. they have inserted recently and have not yet had time to degenerate.THE BIG QUESTION: What protects our genomes from being blasted to bits by retroposition of SINEs and LINEs? The answer appears to be that RNAi is our DNA genome’s immune system!
Six easy steps to understanding RNAi
Step One. A perfect RNA duplex is usually a sign of danger.
RNA
RNA
viral DNA genome
bidirectional transcription of a compact viral genome
Alu RNA
Alu RNA
genomic DNA
transcription of dispersed repeated retroelements ("genomic parasites")
Alu geneL1 L1
L1 RNA
Alu and L1 RNA
Alu Alu
Six easy steps to understanding RNAi
Step Two. The RNA duplex is "diced" into 21 bp fragments.
RNA
RNA
"dicer"-likeenzymes
21 bp siRNAs
long RNAs
Six easy steps to understanding RNAi
Step Three. One RNA strand associates with a RISC-like complex
(RISC = RNA-Induced Silencing Complex)
RNA
RNA
"dicer"-likeenzymes
21 bp duplex siRNAs
long duplex RNAs
one RNA strand associates with a RISC-like protein
complex
21 base siRNAsin active
complexes
RISC RISC RISC RISC RISC
5’
RNApolymerase
DNA
nascent RNA
nucleus
cytoplasm
5’ AAAA 3’
mature mRNA
RISC
RISC-like
nucleosomes mRNA attenuation,
degradation, or even activation
by miRNAs
chromatin silencingby siRNAs and
piRNAs
Step Four. A RISC-like complex anneals with complementary RNA, regulating or degrading the RNA and/or silencing the corresponding
chromatin.
Six easy steps to understanding RNAi
Step Five. Our genome encodes >1200 regulatory microRNAs.
mRNA
cap AAAAAORF
imperfectmatch
translationalattenuation
(usually)
unprocessed microRNA (miRNA)
transcript
1. miRNA excisionby "dicer"-like
enzymes
2. assembly of the miRNAinto a RISC-like complex
RISCDrosha
Dicer
Guo et al. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835-840; Friedman et al (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19, 92-105; Bartel (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.
Six easy steps to understanding RNAi
Step Six. Match with mRNA determines degradation or attenuation.
mRNA
cap AAAAAORF
RISC mRNA
cap AAAAAORF
imperfectmatch
attenuation
perfectmatch
degradation
RISC
Six easy steps to understanding RNAi…
Step One. A perfect RNA duplex is usually a sign of danger.
RNA
RNA
viral DNA genome
bidirectional transcription of a compact viral genome
Alu RNA
Alu RNA
genomic DNA
transcription of dispersed repeated retroelements ("genomic parasites")
Alu geneL1 L1
L1 RNA
Alu Alu
Alu and L1 RNA
5’
RNApolymerase
DNA
nascent RNA
nucleus
cytoplasm
5’ AAAA 3’
mature mRNA
RISC
RISC-like
nucleosomes mRNA attenuation,
degradation, or even activation
by miRNAs
Step Four. A RISC-like complex anneals with complementary RNA, regulating or degrading the RNA and/or silencing the corresponding
chromatin.
chromatin silencingby siRNAs and
piRNAs
de novo silencing maintenance of silencing
transcription
dsRNA formation
RNAi-mediated processing
recruitment of histone methyltransferase
local histone H3K9 methylation
recruitment of chromodomain proteins
Dernburg and Karpen (2002) Cell 111, 159-162; Coulmenares et al. (2007) Mol Cell 27, 449-461
RNAi amplification by RNA-dependent RNA
polymerase
(worms not humans)
DNA replication,proteins dissociate,
histones rebind locally
proteins rebind to methylated histones
methylation spreads, DNA resilenced
chromodomain protein
histone methyltransferase
methylatedhistone H3
KEY
a
b
c
d
Wang et al. (2008) Nature 456, 209-213
Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand
The DNA (red) is bound in a deep channel where the sugar-phosphate backbone is held ina helical conformation and the Watson/Crick bases are stacked and ready for base pairing...thus Argonaute proteins are Nature's own way of making "locked nucleic acids" (LNAs).
()
Here is Herpesvirus saimiri HSUR1 titrating out host miRNAs miR-142-3p and miR-27a
Cazalla et al. (2011) A Primate Herpesvirus Uses the Integrator Complex to Generate Viral MicroRNAs. Mol Cell 43, 982.
So, naturalists observe, a fleaHas smaller fleas that on him prey;And these have smaller still to bite‘em;And so proceed ad infinitum.
Jonathan Swift (1667–1745)Poetry, A Rhapsody
coding ceRNA(competing
endogenous RNA)
regulated mRNA
noncoding ceRNAs(aka lncRNA for long noncoding RNA) may bind miRNAs to linear sequences or multiple
proteins to 3D structures
3’ UTRmiRNA
cap AAAAAA
cap AAAAAA
cap AAAAAAORF
ORF3’ UTR
cap AAAAAA
Molecular circuitry by which lnc-MD1, miR-135, and miR-133 regulate muscle
differentiation
Cesana et al. (2011) A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell
147, 358-369
lnc-MD1
AnAn
microRNAs repress muscle mRNAs through 3' UTR lnc-MD1 RNA relieves
repression by soaking up miRNAs
lnc-MD1
So, naturalists observe, a fleaHas smaller fleas that on him prey;And these have smaller still to bite‘em;And so proceed ad infinitum.
Jonathan Swift (1667–1745)Poetry, A Rhapsody
coding ceRNA(competing
endogenous RNA)
regulated mRNA
noncoding ceRNAs(aka lncRNA for long noncoding RNA) may bind miRNAs to linear sequences or multiple
proteins to 3D structures
3’ UTRmiRNA
cap AAAAAA
cap AAAAAA
cap AAAAAAORF
ORF3’ UTR
cap AAAAAA
A model for lncRNA (long noncoding RNA) function in cellular circuitry. (left) Embryonic Stem (ES) cell-specific transcription factors (such as Oct4, Sox2 and Nanog) bind to the lncRNA promoter and drive transcription. The lncRNA binds to ubiquitous regulatory proteins, giving rise to cell-type specific RNA-protein complexes. Through different combinations of protein interactions, the lncRNA-protein complex gives rise to unique transcriptional programs. (right) A similar process may work in other cell types with specific transcription factors regulating lncRNAs, which form cell type-specific RNA-protein complexes that regulate cell-type-specific gene expression programs.
lncRNAs act in the circuitry controlling pluripotency and
differentiation [Guttman et al. (2011) Nature 477, 295]
aspirin, 180 Da
ibuprofen, 206 Da
The monomer unit of RNA is a 330 Da ribonucleotide; an RNA polymer of 15 monomer units is called an oligonucleotide (or “15-mer”) and would be 5,000 Da
CH3
Why RNA doesn't look "druggable" at first glance
PPPPPP PPP PPPP
PPPPPP PPP PPPP
unmodifiedRNA or DNA
membranebilayer
polyanions cannot penetrate the negatively charged membrane bilayer
Why unmodified oligonucleotides are not good drugs
X
P–P
–P–P
–P–P
–P
P–P
–P–P
–P–P
–P
– –– –– –– –– –– –
PP
PP
PP
PP
PP
cellulartarget RNA
antisenseoligonucleotide
P–P–P–P–P–P–P
cap AAAAAmRNA ORF
The antisense specificity problem is huge: there is no escape from "off-target" effects
cap AAAAAmRNA ORF
cap AAAAAmRNA ORF
cap AAAAAmRNA ORF
cap AAAAAmRNA ORF
cap AAAAAmRNA ORF
oligo too short, binds too weakly
oligo too long, bindsto unintended targets("off-target effects")
"magic bullet" may not exist, biologyis too complicated!
H
H
DNA with phosphorothioate linkages
RNA with phosphorothioate linkages
OH
OH
RNA with phosphorothioate linkages and 2'-O-methyl
modifications
OCH3
O
CH3
siRNAs that cannot be digested by exonucleases and endonucleases, and/or cannot cleave themselves
What is a peptide-nucleic acid or PNA? The neutral peptide backbone in peptide-nucleic acid (PNA) replaces the negatively charged sugar-phosphate backbone of RNA and DNA.
The resulting PNA:RNA or PNA:DNA duplex is much more stable than a pure RNA or DNA duplex, because there is no charge/charge repulsion between the two strands.
PNA is also resistant to degradation by cellular nucleases and proteases.
proteinDNA(or RNA)PNA
peptide bond
Koppelhus and Nielsen (2003) Adv Drug Deliv Rev 55, 267-280;Ivanova et al. (2008) Nucl Acids Res 3, 6418-6428.
LNAs ("locked nucleic acids") enable shorter RNAsto achieve specificity and resist cellular nucleasesA locked nucleic acid (LNA) is an RNA
monomer in which a bridge connecting the 2' oxygen and 4' carbon locks the ribose sugar into the conformation it would normally adopt in an RNA double helix. This conformation enhances base stacking, backbone pre-organization, and increases the melting temperature of the duplex; however, it also prevents LNAs from functioning as a ribozymes. LNA oligomers can be synthesized chemically.
In a therapeutic proof-of-principle, cardiac function and survival both improved when LNA oligomers complementary to miR-208a were injected into the tail vein of hypertensive rats [Montgomery et al. (2011) Circulation 124, 1537].
baseC
locked nucleic acid (LNA)
baseC5'
3'
H2'
4'
RNA monomerin double helix