Brief introduction to non- protein-coding RNAs · 2013-06-28 · miRNA , siRNA , rasiRNA biogenesis...
Transcript of Brief introduction to non- protein-coding RNAs · 2013-06-28 · miRNA , siRNA , rasiRNA biogenesis...
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Brief introduction to non-
protein-coding RNAs
Mihaela Zavolan
Biozentrum, Basel
Swiss Institute of Bioinformatics
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Classes of functional RNAs
• rRNA - protein synthesis
• tRNA - transport of amino acids
• snRNA - spliceosome
• snoRNA - rRNA methylation and pseudouridylation
• RNase P - removal of 5’ sequence from tRNAs
• SRP RNA - protein secretion pathway
• miRNA,siRNA - translation inhibition / mRNA degradation
• antisense RNAs (Xist) - X chromosome inactivation
• bacterial noncoding RNAs
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From genes to proteins
pre-mRNA
Transcription
Splicing, capping, polyadenylation
mature mRNA
cDNA
genome
Start Stop
Capture, cDNA synthesis Translation
protein
) ) )( ( (
m3G A AA
AA
Exon 1 Exon 2 Exon 3 Exon 4
5’ splice site )3’ splice site (
) )
)
Start
) )) )( ( (
Stop
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snRNA
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tRNA
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rRNA
70 S
2.5x106 Da
50 S
1.6x106 Da 30 S
0.9x106 Da
5 S rRNA
120 nc
23 S rRNA
2900 nc
16 S rRNA
1540 nc
34 proteins 21 proteins
80 S
4.2x106 Da
60 S
2.8x106 Da 40 S
1.4x106 Da
5 S rRNA
120 nc
28 S rRNA
4700 nc
18 S rRNA
1900 nc
~49 proteins
~33 proteins
5.8 S rRNA
160 nc
Prokaryotic Eukaryotic
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rRNA
RNA - gray
Peptide - gold backbone
Large ribosomal subunit of Haloarcula mortui - Ban et al. Science 289:905-920 (2000)
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snoRNA
Weinstein & Steitz, 1999
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snoRNA
Cavaille et al. 2002
Cavaille et al. 2000
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RNAi: why so much excitement?
• Unsuspected, intricate regulatory pathways
• Involving many new enzymes and RNA-binding proteins
• Regulating a large fraction of the genes
• Extremely versatile tool to knock down gene expression
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Short history of RNAi
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
• 1995 - Guo & Kemphues discover RNA interference
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
• 1995 - Guo & Kemphues discover RNA interference
• 2000 - The group of G. Ruvkun discovers the first
evolutionarily conserved microRNA (let-7) in the worm
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
• 1995 - Guo & Kemphues discover RNA interference
• 2000 - The group of G. Ruvkun discovers the first
evolutionarily conserved microRNA (let-7) in the worm
• 2001 - The group of T. Tuschl shows that RNAi
is mediated by small (21-22 nc) RNAs
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
• 1995 - Guo & Kemphues discover RNA interference
• 2000 - The group of G. Ruvkun discovers the first
evolutionarily conserved microRNA (let-7) in the worm
• 2001 - The group of T. Tuschl shows that RNAi
is mediated by small (21-22 nc) RNAs
• 2002 - Hutvagner & Zamore show that miRNAs and
siRNAs share components of the effector pathway
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Short history of RNAi• 1990 - multiple copies of a pigment transgene result in
colorless petunias (Napoli et al.)
• 1993 - The group of V. Ambros discovers the first
microRNA (lin-4) in the worm
• 1995 - Guo & Kemphues discover RNA interference
• 2000 - The group of G. Ruvkun discovers the first
evolutionarily conserved microRNA (let-7) in the worm
• 2001 - The group of T. Tuschl shows that RNAi
is mediated by small (21-22 nc) RNAs
• 2002 - Hutvagner & Zamore show that miRNAs and
siRNAs share components of the effector pathway
• 2004 - RITS complex purified by the group of D. Moazed
• 2005 - hundreds on miRNA genes in human (321),
mouse (245), rat (194), chicken (122), fly (78),
worm (114); hundreds more predicted.
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miRNA, siRNA, rasiRNA biogenesis
dsRNA
DICER
Exogenous dsRNA
• Viral infection
• Transfection
Endogenous dsRNA
• Repetitive elements
• Antisense transcripts
RISC
mRNA
mRNA cleavage
siRNA
MicroRNA precursors
encoded in the genome
DICER
AAAA3’-UTR
miRNA
RISC-like
miRNP
RNA interference pathway MicroRNA pathway
Me Me Me Me
Chromatin silencing Translational repression
RITS
rasiRNA
Drosha
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Biological functions of RNAi/miRNAs
• Defense mechanisms against foreign nucleic acids
- Viral infection
- Active transposons
• Regulation of gene expression
- globally (formation of heterochromatin, centromers)
- locally (stellate locus silencing)
- translational regulation by miRNAs
- transcriptional regulation by miRNAs (plants)
• Regulation of genomic rearrangement (Tetrahymena, Paramecium)
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Proteins involved in RNAi function
Components of RISC and miRNPs
Paz (siRNA-) Piwi (Dicer-binding) domain family (Ago, Hiwi)
Fragile X Mental Retardation Protein (FMRP) and related proteins
VIG, Gemins, Tudor-SN, R2D2, Dicer
Other proteins RNA-dependent RNA polymerases (RdRPs)
RNA helicases
Proteins modulating RNAi (eri-1)
Viral supressors of RNAi
Various genes of unknown function identified by screening
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Main problems with RNAi technology
siRNAs
RISC
mRNACleavage
AAAA
3’-UTR
RISC-like
miRNP
Off-target effects
Delivery of siRNAs to the appropriate tissue
Sustainability of the inhibitory effect
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Specificity of RNAi/miRNAs
5’ end of the miRNA is critical for miRNA
function. Doench & Sharp 2004.
RISC
mRNA
mRNA cleavage
Ago2
Ago2 is a cleaving endonuclease.
Joshua-Tor & Hannon 2004
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Applications of RNAi
High-throughput RNAi screens for elucidating gene function
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Applications of RNAi
Development of RNA-
based anti-viral
(HIV1, Influenza,
Coxsackie, HCV)
and anti-cancer
(targets:
angiogenesis, DNA
repair, STAT3, bcl-2)
therapeutics
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Functions of miRNAs
Let-7 miRNA regulates
developmental timing in
C.elegans. Reinhart et al. 2000
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Functions of miRNAs
Ras is regulated by let-7 miRNA.
Johnson et al. 2005
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Functions of miRNAs
Pancreatic-specific miR-375
inhibits insulin secretion.
Poy et al. 2004
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Functions of miRNAs
miRNAs regulate brain
morphogenesis in zebrafish.
Giraldez et al. 2005
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rasiRNAs in epigenetic regulation
Heterochromatic RITS-associated siRNAs in fission
yeast originate from diverse DNA elements.
Cam et al. 2005.
Not demonstrated
yet in mammals.
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Some ncRNA databases
• general: ncRNA database
http://noncode.bioinfo.org.cn/
• microRNA repository
http://microrna.sanger.ac.uk/sequences/
• tRNA databases http://lowelab.ucsc.edu/GtRNAdb/
http://medstat.med.utah.edu/RNAmods/trnabase/
http://www.staff.uni-bayreuth.de/~btc914/search/
• rRNA database http://www.psb.ugent.be/rRNA/