structural RNAs!
sRNAs!
siRNA!
miRNA!
lncRNAs!
pervasive
transcripts!
HISTORY OF RNA
Rinn and Chang, Ann. Rev. Biochem, 2012
RNA made via condensation from ribose and other organic substances
RNA evolution- molecules learns to replicate
RNA starts to join aminoacids and synthesises polypeptides and proteins
Proteins aid RNA to replicate and make proteins. dsRNA evolves into stable DNA.
DNA and proteins take over major roles as genetic information and enzymes
„primordial soup”
„prebiotic soup”
„THE RNA WORLD” hypothesis
pre-RNA world RNA world
RNA+proteins
RNA+DNA+
proteins
Library of randomized RNA sequences (1015)
SELEX cycles
1. binding
2. washing
3. elution 4. Amplification
RT-PCR
5. in vitro transcription
final molecules:
cloning, analysis
last cycle
Enriched library
discard -
molecules that do not bind
molecules that bind
EnrichmentTarget
tests
SELEX = Systematic Evolution of Ligands by EXponential enrichment
Szostak Gold
1990
Method of selecting RNA/DNA molecules with desired properties (aptamers, ribozymes) based on cycles of amplification
Selected RNAs:
- cleave DNA i RNA - ligate RNAs
- self-replicate
- create peptide bonds
ncRNA
• Housekeeping
- constitutively expressed
- required for normal function and cell viability
• Regulatory
- expressed temporarily (development, response to stimuli)
- affect gene expression at the level of transcription or translation
• tRNA and rRNA – translation
• snRNA – splicesosome components, pre-mRNA splicing
• snoRNA – rRNA processing and modification, scaRNA (CB specific)
• RNA components of RNase P and RNase MRP – endonucleases:
tRNA and rRNA processing
• Signal Recognition Particle SRP RNA – protein secretion to ER
• tmRNA tRNA-mRNA hybrid- targeting nascent proteins for degradation
• gRNA – guide RNA in RNA editing
• telomerase RNA – synthesis of telomers
• sRNAs: siRNA (exo-siRNAs and endo-siRNAs; ta-siRNA; nat-siRNA;
lsiRNAs); miRNA; piRNA – act in TGS or PTGS
• lncRNAs – much less known, usually act in TGS (chromatin level)
Tisseur et al., Biochemie, 2011
ALL
ncRNAs?
GENE SILENCING – RNAi
DISCOVERY OF 2002:!
ncRNAs in RNAi!
siRNAs/miRNAs:
- double stranded small noncoding RNAs
- complementary to mRNA targets
- participate in gene silencing
- mediate:
TRANSCRIPTIONAL GENE SILENCING (TGS)
• transcription inhibition POST-TRANSCRIPTIONAL GENE SILENCING (PTGS)
• mRNA cleavage or
• translation inhibition or
• translation activation
Rusk, Nature Meth, 2008
Activation of translation (PTGS)
Interfering RNAs: mode of action
Tijsterman and Plasterk, Cell, 2004
miRNA siRNA
siRNA/miRNA-dependent cleavage (PTGS)
Inhibition of translation (PTGS)
Transcriptional silencing (TGS)
Bartel, Cell, 2004
PTGS : Post Transcriptional Gene Silencing (RISC)
mRNA cleavage or mRNA decay, translation repression /activation
• siRNA (exo-siRNAs and endo-siRNAs; ta-siRNA; nat-siRNA; lsiRNAs)
• miRNA (mirtron)
• piRNA!
TGS : Transcriptional Gene Silencing (RITS)
heterochromatin formation, DNA methylation, transposon silencing
• siRNA!
• piRNAs !
Holo-RISC
RNA Pol RNA Pol IV
PTGS : Post Transcriptional Gene Silencing (RISC)
mRNA cleavage or mRNA decay, translation repression/activation
• siRNA, miRNA- ( plants, algae, animals, viruses, protists ); piRNA (metazoans)
• exo-siRNAs – exogenous siRNA, act usually in cis (animals, plants, fungi)!
viral-siRNAs – exogenous siRNAs, viral genome (plants)
transgene siRNAs – against foreign RNA (animals, plants, fungi)!
• endo-siRNAs - endogenous siRNAs, act usually in cis !
• ta-siRNA – trans-acting, generated by miRNA cleavage of ncRNAs (plants)!
• nat-siRNA- natural antisense-derived siRNAs (plants)!
• lsiRNAs – long siRNAs (30–40 nts) derived from antisense RNAs (plants)
• mirtron- intron-encoded miRNAs that bypass Dicer processing (animal)!
miRNA
(microRNA)
plants animals viruses, Protista
20–25nt
Drosha
(animals)
+ Dicer
Pol II /Pol III transcripts
mRNA stability
translation inhibition (mRNA cleavage or decay)
mirtrons – derived from pre-mRNA introns; present in animals
siRNA
(small interferring RNA) – most act in cis, except tasiRNA
exo-siRNA
(exogenous)
plants fungi animals Protista
21-24nt Dicer transgenic, virus-derived or other exogenic RNA
PTGS
anti-virus defence
endo-siRNA
(endogenous)
plants fungi, animals Protista
~21nt Dicer
Bi-directional or
convergent transcription mRNA base-pairing with antisense pseudogenes
PTGS,
transposon regulation
tasiRNA
(trans-acting siRNA)
plants 21nt DCL4 TAS RNA precursors
cleaved by miRNA PTGS
natsiRNA
(natural antisense transcripts- derived siRNA)
plants 24nt 21nt
DCL2 DCL1
Stress-induced
bi-directional transcription Stress response
PTGS : Post Transcriptional Gene Silencing (RISC)
TGS : Transcriptional Gene Silencing (RITS)
siRNA
(small interferring RNA)
endo-siRNA
(endogenous)
plants fungi animals Protista
~21nt Dicer
Bi-directional or convergent transcription
mRNA base-pairing with antisense pseudogenes
TGS,
transposon regulation
hc-siRNA
(repeat-associated heterochromatic siRNA)
plants
S. pombe 24-26nt DCL3 Transposons, repeats Chromatin modification
piRNA
(Piwi-interacting RNA)
Drosophila, C. elegans, mammals, Danio rerio
24–30nt Dicer-
independent Long primary transcripts transposon regulation
heterochromatin formation, DNA methylation, transposon silencing
RNA Pol IV-V and RDRP- DNA/RNA dep RNA Pol involved in siRNA
production and amplification (plants, worms)
DICER and AGO:
two major players
PAZ
RIII
RIII
DICER or DICER-like (DCL)
Liu and Paroo, Annu. Rev. Biochem., 2010 DICER
dsRNA
siRNA
RISC
AGO
DICER and AGO:
two major players
Liu and Paroo, Annu. Rev. Biochem., 2010
ARGONAUTE
Plants
A. thaliana 10 - 4 (DCL 1-4) 6
O. sativa 18 - 5 5
S. cerevisiae - - - -
S. pombe 1 - 1 1
N. crassa 1 - 1 3
A. nidulans 1 - 1 2
C. elegans 5 3 2 (Dicer + Drosha) 4 D. melanogaster 2 3 3 (2 Dicers + Drosha) -
D. rerio 4 4 2 (Dicer + Drosha) -
H. sapiens 4 4 2 (Dicer + Drosha) -
Multiple DICERs and AGOs
DCL4 DCL1
Czech and Hannon, Nat. Rev. Genet., 2010
siRNAs
viral ssRNA
viral RdRP
AGO1/2 21-22nt siRNA
AGO1/2 silencing of viral
replication viral dsRNA
plants
metazoans
AGO1/2 21-22nt
siRNA mRNA cleavage
exogenous dsRNA
AGO1/2
RdRP
siRNA amplification
secondary siRNA AGO
Signal amplification
siRNAs in TGS (plants)
AGO4
siRNA
DCL3
RDR2
2°siRNA
RNA Pl IV RNA Pol IV
DCL3 RNA Pol V
Pol IV/Pol V (plants)
Wierzbicki, Current Opinion Plant Biol., 2012
• lncRNA precursors to siRNA generated by Pol IV recruited by SHH1, CLSY1 provides processivity!
• dsRNA precursors produced by PolII
(overlapping or inverted repeat transcripts)!
• RDR2 co-transcriptionally converts lncRNAs into dsRNAs
• 24-nt siRNAs produced by DCL3 (Dicer)
• Pol V synthesizes scaffold transcripts
RDM1, DRD1, DMS3, DMS11 provide processivity!
• Pol V transcripts bind AGO4/siRNA, SPT5L, IDN2
• AGO4/siRNA recognizes genomic targets by basepairing with lncRNA (PolV)
• AGO4 interacts with WG/GW domains of Pol IV and SPT5L
• transcriptional silencing is established by de
novo DNA methylation by DRM2
Law and Jacobsen , Nat Rev Genet, 2010
siRNAs in TGS (plants)
Shiv et al., Nature., 2007
siRNAs in TGS (S. pombe)
miRNAs
Gangaraju and Lin, Nat. Rev. Mol.Cel. Biol., 2009
Voinnet, Cell, 2009; Ghildiyal and Zamore, Nat. Rev. Genet., 2009
Ago-associated 20–24 nts Dicer-dep., dsRNAs
Ago-associated 21 nts, dsRNAs plant specific miRNA-dep.
tasiRNAs…
siRNAs
Piwi-associated 26–32 nts, ssRNAs germline specific Dicer-indep.
piRNAs
miRNA BIOGENESIS (animals)
Krol et al., Nat.Rev.Genet., 2010
Jones-Rhoades et al., Ann.Rev.Plant.Biol.., 2006
miRNA BIOGENESIS
(plants)
siRNA/miRNA-dependent RNA decay
stress, development
Houseley and Tollervey, Cell, 2009
MECHANISM of miRNA-mediated PTGS (plants)
MECHANISM of miRNA-mediated PTGS (animals)
TRANSLATIONAL REPRESSION
Step 1. Initial effect of miRNAs: inhibition of translation at the initiation step without mRNA decay.
Step 2. mRNA deadenylation by PAN2–PAN3 and CCR4–NOT complexes recruited by
miRISC as a consequence of translation inhibition that makes poly(A) tail more accessible.
Step3. Stimulated deadenylation potentiates the effect on translational inhibition and leads to decay of target mRNAs through the recruitment of the decapping machinery
Destabilization of target mRNA is the predominant reason for reduced protein output.
Liu et al, Nature Cell Biol, 2005 Parker and Seth, Mol. Cell, 2007
P bodies, GW bodies, siRNAi bodies
Jouannet et al., EMBO J, 2012
REGULATION of miRNAs
Krol et al., Nat.Rev.Genet., 2010
ncRNA regulation
• ncRNAs control gene expression (mRNAs) on different levels during various cellular and development processes
• expression of ncRNAs itself is regulated via transcription, biogenesis, protein binding and turnover
Ding et al, TiB, 2009
LIN28/28B binds to let7 family pri-miRNAs and block their processing by Drosha in the nucleus
LIN28 binds to let7 pre-miRNAs and block their processing by Dicer in the cytoplasm
let7 miR-128 targets lin-28 mRNA 3’ UTR inhibiting LIN28 translation!
miRNA degradation
3’ oligouridylation
PUP Poly(U) Polymerases TUTase Terminal Uridylyl Transferase
mature!
Arabidopsis !
Chlamydomonas!
Krol et al., Nat.Rev.Genet, 2010; Kai and Pasquinelli, Nat.Str.Mol.Biol., 2010; Krol et al., Cell, 2010
precursors C. elegans!
RNAi and EDITING
Nishikura,Ann. Rev. Biochem., 2010
Ota et al., Cell 2013
Additional function of ADAR1:
ADAR1 increases the rate of pre-miRNA cleavage by Dicer and facilitates loading of miRNA onto RISC complex
RNAi and EDITING
ADAR1 homodimer – editing
ADAR1/Dicer dimer - silencing
Xhemalce et al., Cell, 2012
miRNA regulation via modification
BCDIN3D methyltransferase phospho-dimethylates 5’ end of pre-miRNA, impairing its
processing by Dicer
Heo et al., Cell, 2012
Drosha processing of group II miRNA (let-7b) gives 1 nt 3’ overhang Oligo-uridylation in the presence of let-7 suppressor Lin28 (embryonic stem cells) by TUT4 leads to miRNA decay.
Mono-uridylation in the absence of Lin28 (somatic cells) by TUT7, 4 or 2
results in miRNA processing by Dicer
miRNA FUNCTION (plants)
miRNA FUNCTION (animals)
C. elegans
development lin-14 gene
miRNA FUNCTION (animals)
Mercer et al., Nat.Rev.Genet., 2007
ncRNA recruit chromatin modifying complex to genes, resulting in H3meK27 and heterochromatin formation
ncRNAs act as repressors or enhancers of
transcription via binding to protein factors or DNA
ncRNAs mask 5’ splice
site resulting in intron
retention, recognition of
IRE and translation
FUNCTIONS of LONG ncRNAs
Chen and Carmichael, WIRERNA, 2010
FUNCTIONS of LONG ncRNAs
Wapinski and Chang, TiCS, 2011
MECHANISM of ACTION of LONG ncRNAs
Nagano and Fraser, Cell, 2011
Cotranscriptional recruitment of chromatin-modifying factors.
Nucleation of chromatin.
Dynamic assembly of nuclear structures:
paraspecles, nuclear bodies
Formation of higher-order chromatin loops
• GUIDES (chromatin modifyiers)
• TRX FACTORS
• SCAFFOLDS (RNP structures)
Xist ncRNA – inactivation of X chromosome
Xist (X-inactive specific transcript, 19 kb ) expressed from inactive X
wrapped around X
Tsix (40 kb) expressed from active X
Dosage compensation – one copy of X chromosome
in females is epigenetically silenced (mammals)
FUNCTIONS of ANTISENSE RNAs
Rosonina and Manley, Dev. Cell, 2010
FLC – Flowering Locus C
- MADS box transcription factor - major repressor of flowering in plants
- expression regulated by FLC antisense
• two major forms of FLC antisense are synthesized
• regulated by alternative polyadenylation by RNA binding proteins, FPA and FCA, and CTSF factor FY
• short asFLC (3’ processing at site I) recruits histone demethylase FLD which introduces transcriptionally repressive histone modifications leading to FLC silencing
• long asFLC (3’ processing at site II) causes nucleosomal rearrangements at
the FLC promoter leading to FLC transcription
MALAT1/mascRNA
• Pol II polyadenylated transcript, a minor form of MALAT1,
precursor to mature MALAT1 and mascRNA
• Processing by RNAseP (5’) and RNaseZ (3’) releases 6.7 kb
MALAT1 and tRNA-like
mascRNA, exported to cytoplasm after addition of the CCA
Wilusz and Spector, RNA, 2010
MALAT1:
- metastasis-associated lung adenocarcinoma transcript 1 (NEAT2 in humans) - enriched in nuclear speckles
- possibly regulates alternative splicing (associates with SR proteins) mascRNA:
- present in the cytoplasm, processed from pre-MALAT1, function unknown
NEAT1 and MALAT1 !
NEAT1 MALAT1
paraspecles nuclear
specles
Chen and Carmichael, WIRERNA, 2010
TERRA – telomeric repeat-containing RNA
(yeast and human)
Luke and Lingner., EMBO J, 2009
• polyadenylated Pol II transcript
• spans subtelomeric and telomeric regions
• a component of telomeric heterochromatin
• associates with telomeres and telomere proteins (Trf1, Trf2)
• regulated by RNA surveillance ( Rat1 , Trf4, NMD factors, RNAse H)
• regulates telomerase (telomere shortening) via RNA-DNA hybrids
• acts in chromatin remodelling (development and differentiation)
• affects telomere replication
• upregulated in ICF patients
(Immunodeficiency, Centromeric region
instability, Facial anomalies)
Chen and Carmichael, WIRERNA, 2010
INVISIBLE RNAs
PERVASIVE TRANSCRIPTION OF THE GENOME
(1) protein-coding mRNA; (2) PROMPT - promoter upstream transcripts (short); (3) PASR- promoter-associated sRNAs (< 200 nts); (4) TSSa
transcription start site-associated RNAs (20-90 nts); (5) TASR –terminator associated sRNAs (< 200 nts); (6) PARL - promoter-associated long RNAs (> 200 nts); (7) tiRNAs - tiny transcription-initiation RNAs (18 nts)
SAGE, CAGE, GRO tags
antisense RNAs (can be long)
CUTs, SUTs - cryptic unstable or stable unannotated transcripts (200-600 nts)
Jacquier, Nat.Rev.Genet., 2009
CUTs, SUTs, XUTs, MUTs and ALL THAT JAZZ
CUT = Cryptic Unstable Transcripts SUT = Stable Unannotated Transcripts SAT = Ssu72-associated Transcripts
XUT = Xrn1-dependent UnstableTranscripts MUT = Meiotic Unstable Transcripts
NO LONGER
TRANSCRIPTIONAL NOISE
(yeast, mammals, worms, plants - all organisms?)
• not visible in normal wild-type cells
• accumulate in RNA degradation mutants (EXOSOME, XRN family, TRAMP) or various metabolic conditions (aging, nutrient change, cell cycle etc)
• originate from widespread bidirectional promoters
• „mRNA-like” Pol II transcripts (capped, polyadenylated)
Jacquier, Nat. Rev. Genet., 2009
Tisseur et al., Biochemie, 2011
GENOMIC ORGANIZATION of ncRNA
short
long
Wyers et al., Cell, 2005; Arigo et al., Mol.Cell, 2006a; Thiebaut et al., Mol.Cell, 2006, 2008; Houseley et al., EMBO J, 2007; Camblong et al., Cell, 2007; Thompson and Parker, Mol.Cell. Biol., 2007; Houseley et al., Mol. Cell, 2008; Vasiljeva et al., Mol.Cell, 2008; Luke et al., Mol. Cell, 2008;
Berretta et al., Gene Dev., 2008; Preker et al., Science, 2008; Seila et al., Science, 2008; Xu et al., Nature, 2009; Neil et al., Nature, 2009
ncRNA instability and their termination mode
Unstable CUTs
- are detected in TRAMP or exosome mutants
- are terminated by Nrd1/Nab3-dependent mechanism and polyadenylated by Trf4/TRAMP
- Nrd1/Nab3, TRAMP and exosome complexes interact - some CUTs (SRG1, IGS1-R) are polyadenylated by Pap1
- some CUTs are exported to the cytoplasm (XUTs) and degraded by Xrn1
- ncRNP composition is largely unknown
PHYSIOLOGICAL FUNCTIONS of CUTs
Camblong et al., Cell, 2007; Wery et al., WIREsSMB’11
Similar PHO84 silencing occurs in aging yeast
Stabilization of as CUT leads to H3K18 deacetylation by Hda1 at PHO84 promoter Regulation of gene expression via antisense RNA and epigenetic modification:
PHO84 (inorganic phosphate transporter)!
PHYSIOLOGICAL FUNCTIONS of XUTs
Transcriptional silencing of the Ty1 transposon
Berretta et al., Gene Dev, 2008; Wery et al., WIREsSMB’11
directly or indirectly controlled by Set1
antisense TY1 XUT
• polyadenylated Pol II transcript
• antisense to TY1 promoter
• degraded by cytoplasmic Xrn1
• silences TY1 expression by promoting histone deacetylation and trimethylation (by Set1)
• can act in-trans
Berretta and Morillon, Embo Rep. 2009
CUT transcribed in-cis, when stabilized, recruits chromatin modification enzymes (HDAC) to gene promoter
CUT transcribed from a distant locus, when stabilized, recruits chromatin modification enzymes (HTM) to inhibit transcrition
CUT ACTION in-cis or in-trans!
UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments
Thompson and Parker, Cell, 2009
Stress-induced enzymatic tRNA cleavage
(S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines)
Selected agiogenin-derived 5’-tiRNAs with terminal oligoG at the 5’end
• repress translation in vitro and in vivo
• displace eIF4G/eIF4A from uncapped transcripts and eIF4F from m7G cap
• trigger formation of stress granules (SGs)
• translational repressor YB-1 contributes to tiRNA mediated repression
Ivanov et al, Mol Cell, 2011
UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments
• > 17 short abundant tRFs (13–26 nts), generated by RNaseZ from mature (5’ and 3’ ends) and precursor (3’ trailer) tRNAs identified in the cytoplasm in prostate cancer cells. Lack of tRF1001 impairs cell proliferation.
• Abundant Dicer-dependent tRFs (class I, from mature 3’ and 5’ ends) in HeLa moderately downregulate target genes.
• Class II tRFs (from RNAseZ 3’ cleavage to Pol III termination, cytoplasmic) associate with Ago2–3. Function- regulation of silencing via differential
association with Ago proteins?
Haussecker et al., RNA, 2010 Cole et al., RNA, 2009
Lee et al., Gene Dev., 2009
TAKE-HOME MESSAGE
• ncRNAs have house-keeping (translation, splicing, protein sorting, RNA processing) and regulatory functions
• ncRNAs affect processes such as transcription (chromatin structure), RNA stability and translation
• The majority of eukaryotic genomes are transcribed giving rise to a variety of RNAs
• At least some of the “invisible” transcripts in some conditions form functional ncRNAs
• These usually act in transcriptional silencing in-cis or in-trans by recruiting chromatin modifying enzymes
• Defects in ncRNA level or activity correlate with
several diseases
rDNA silencing by pRNA and NoRC
Matthews and Olsen, Embo Rep., 2006;
Tucker et al., Cur. Op. Cell. Biol., 2010
NoRC – mammalian nucleolar remodeling complex which establishes and maintains heterochromatic state at
promoters of silent rDNA repeats (histone modifications and CpG methylation)
- TIP5 TTF-I-interaction protein5 - SnF2 ATP-dependent chromatin remodeler
other
- TTF-1 transcription factor I
- UBF upstream binding factor
- DNMT DNA methyltransferase
- HDAC1 histone deacetylase
rDNA silencing by pRNA and NoRC
ADDITIONAL SILENCING
• pRNA binds at T
0to rDNA promoter, independently of TTF-I and other proteins, forming a triplex
• pRNA competes with TTF-I
• rDNA/pRNA triplex recruits methyltransferase DNMT3b
• this results in chromatin hypermethylation and rDNA silencing
Stark and Taliansky, Embo Rep., 2008; Mayer et al., Mol. Cell, 2006; Embo Rep., 2008; Schmitz et al., Gene Dev., 2010
• NoRC function requires TIP5 association with pRNA
• NoRC brings DNA and
histone modifying enzymes leading to hetrochromatin formation
• CpG-133 methylation prevents binding of UBF
• this inhibits formation of the transcription complex
pRNA processed to
Pol I intergenic
transcript
Kai and Pasquinelli, Nat.Str.Mol.Biol,2010
