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