rna silencing
TRANSCRIPT
RNA SilencingRNAi
Post transcriptional gene regulation by siRNA and miRNA
Pete Burrows
MIC 759
October 26, 2006
Small untranslated regulatory RNAsEukaryotes ncRNAProkaryotes sRNA
Guillier, et al. Genes and Development 20:2338, 2006
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Entrez PubMed Search Terms: RNAi or siRNA or miRNA
Number of Publications
Lecture Outline
• Discovery
• General features
• siRNA– Role of translation
• miRNA
• Problems
Focus on RNA interference - A user’s guide
September 2006Nature Genetics
June 2006 Supplement
Early demonstration of RNAi in plants
In situ hybridization for mex-3 mRNA 4 cell embryos
No probe
anti-sense ssRNA
No RNA
dsRNA
391:806, 1998
Nature 391:744 1998
News and Views
In vitro transcription
Restriction digest
Blunt end
5’ overhang3’ overhang
RNAiDicing and slicing
• RNA silencing pathways are triggered by 21-27 nt long small RNAs– Small interfering RNAs – siRNA– Repeat-associated small interfering RNAs –rasi RNAs– Micro RNAs – miRNA– Piwi-interacting RNA - piRNA
• RNAi induction using long dsRNA only operates in plants and invertebrates
• Worms – soak them in a solution of dsRNA, feed them bacteria expressing the appropriate construct
• In vertebrates, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNase L, degrades mRNA
Novina and Sharp Nature 430:161 2004
Dicer
• Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity
• Fly Dicer requires ATP, human may not
RISC
• RISC has helicase, exonuclease, endonucelase and homology searching proteins.
• Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense (passenger) strand
• Antisense (guide) strand defines specificity of RNAi
Processing of siRNA
• Starting with dsRNA• Which becomes guide strand in the RISC
and which (passenger strand) is excluded? – Sequence and structure– Strand with the less-tightly base pared 5’ end
is incorporated becomes guide strand
• What is the endonuclease (Slicer) in RISC?
The ago1 mutant Arabidopsis develops abnormally because it does not produce aneffector of silencing. The Argonaute genes were so named because the mutant plantslook like an argonaute squid.
The Sainsbury LaboratoryJohn Innes CentreColney LaneNorwich, NR4 7UH, UK
Knew that Ago a RISC component
Published by AAAS
J. Liu et al., Science 305, 1437 -1441 (2004)
Identification of Argonaute 2 as Slicer in humans
Published by AAAS
J. Liu et al., Science 305, 1437 -1441 (2004)
Fig. 2. Argonaute2 is essential for mouse development
Published by AAAS
J. Liu et al., Science 305, 1437 -1441 (2004)
Fig. 3. Argonaute2 is essential for RNAi in MEFs
Sontheimer and Carthew, Science 2004 Sep 3;305(5689):1409-10
Cytosolic players in siRNA and miRNA• Dicer (DCR)
– Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA
• TRBP– TAR RNA Binding Protein, Cofactor for Dicer
• RISC– RNA induced silencing complex
• Argonaute (AGO)– PAZ domain binds the characteristic two-base 3' overhangs of
siRNAs – PIWI domain: dsRNA guided hydrolysis of ssRNA– Ago2 is slicer in mammalian RISC– Other Ago may function in miRNA silencing
Taphrina S. pombe S. cerevisiae Morel Penicillium
http://www.glocalbeer.dkhttp://tolweb.org/tree?group=Ascomycota&contgroup=Fungi
Swahili word for beer (Pombe)
Schizosaccharomyces pombe has DCR and AGO but not in Saccharomyces cerevisiae
Is siRNA coupled to translation?
Shen, et al. Differentiation 73:287-293 2005
Ferritin IRE-IRP
Shen, et al. Differentiation 73:287-293 2005
Shen, et al. Differentiation 73:287-293 2005
Fe + - + - + - + -----Control---- -------IRE-------
siRNA siRNA
7:719, 2005
7:633, 2005
Colocalzation of Ago2 (Slicer) and Dcp1a (Decapping enzyme) in P-bodies
Sen and Blau, The FASEB Journal, 2006 20:1293
RCK human homolog of yeast Dhh 1pInteracts with eIF4e and represses translationTargets RNA to P-bodies?
RNAi
The Movie
Nature Genetics
2006
miRNA
• The miRNA are endogenous small RNA guides that repress the expression of target genes.
• Differ from siRNA in biogenesis not in functions, although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not.
• lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message.
• Lin-14 mRNA levels do not decline, but that may not always be the case.
• let-7 also found in other species.
miRNA• Abundant ssRNA from a few thousand to 40,000
molecules /cell• Found in all metazoans• 0.5-1% of genes• siRNA targets genes from which it is derived in a
sequence specific manner• miRNA regulate separate genes and has imperfect
complementarity• May be 100’s mRNA regulated by one miRNA• Usually have many binding sites in each 3’ UTR,
and several different miRNA can target same 3’ region. Combinatorial control
miRNA
• Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes.
• miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.
Plasterk Cell 124:877, 2006
Du, T. et al. Development 2005;132:4645-4652
The structure of human pri-miRNAs
Processing of miRNA
• Long primary Pol II transcript (pri-miRNA)• Cleaved by Drosha, nuclear RNase III endonuclease to
establish one end of the miRNA (pre-miRNA)– Also need dsRNA binding protein Pasha (flies) DGCR8
(humans)
• The pre-miRNA exported from the nucleus by Exportin 5• Cut by Dicer→ miRNA• Strand with the less-tightly base pared 5’ end becomes
mature miRNA, other strand becomes miRNA* and degraded
• Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each.
Players in miRNA biogenesis
• Drosha– Nuclear RNase III enzyme. Initiates miRAN
biogenesis by cleaving pri-miRNA into pre-miRNA
• Pasha– Partner of drosha is a dsRNA binding protein.
Human DGCR8
• Exportin-5– Nuclear transmembrane protein that transports
pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran
Cullen Nature Immunology 7:563 2006
Du, T. et al. Development 2005;132:4645-4652
The miRNA biogenesis pathway
Mechanism of miRNA suppression of gene expression
• Transcription• mRNA degradation• Translational repression
– 1 Initiation– 2 Elongation– 3 Termination– 4 Release
• Co-translational degradation of the nascent peptide
• Western blot for LIN-14 protein• Transcription same (run-on)• RNA levels ~ same• Lin-4 miRNA expressed at end of L1
Measurement of lin-14 poly(A) tail length
lin-14 RNase protection assay
Polysome Profiles
Polyribosome association of lin-14 mRNA is unchangedbetween L1 and L2
lin-14 RNase protection assay
Polysome Profiles
EDTA sensitivity of polysomes
Proc. Natl. Acad. Sci. USA 102: 16961, 2005
Copyright ©2005 by the National Academy of SciencesHumphreys, David T. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 16961-16966
Fig. 1. miRNAs target the initiation step of translation
No eIFs or Met-tRNAimet
Fátima Gebauer & Matthias W. HentzeNature Reviews Molecular Cell Biology 5, 827-835 (2004)
Scanning model of Cap-dependent Translation Initiation
E P A
E P AeIF5B
Other Studies• Both 5’ cap and 3’ poly(A) tail are
necessary but not sufficient for miRNA repression of translation
• EMCV IRES– Uses everything that canonical translation
initiation does except eIF4E– Resistant to miRNA
• New model– Block in translation initiation– Sequestration in P-bodies– In some cases this may lead to mRNA decay
Is RNAi involved in viral immunity in the vertebrates?● Some viruses are more pathogenic in insects
lacking dicer-2● Viruses encode proteins that inhibit Dicer
– Flock house virus– Greasy grouper nervous necrosis virus
● Does the host cell use RNAi to destroy viral RNA or inhibit its translation?
● Predictions?
Cullen Nature Genetics 38:S25, 2006
In vivo applications of RNAi
• Highly specific– Silence a single nucleotide difference in a dominant
negative allele
• Resistance not (less) a problem– Can design new RNAi if a mutation arises and original
targeted sequence is changed
• Problems– Stability– Delivery– Toxicity
Off Target Effects
• Global, due to induction of innate immune responses
• Cross reactive, due to sequence homology with other mRNA sequences
• Not easy to recognize unless global gene expression studies performed.
• Good to have multiple target sequences
• Recently found in flies
Nature Biotechnology 24:697, 2006