Rnai and gene silencing
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RNAi and Gene Silencing. Pete Burrows MIC 759 September 16, 2008. Lecture Outline. Background/discovery siRNA/shRNA Movie miRNA Biogenesis Functions Applications. # Publications. Focus on RNA interference - A user’s guide September 2006. 1998 Feb 19;391(6669):806-11 . No RNA.

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Rnai and gene silencing

RNAi and Gene Silencing

Pete Burrows

MIC 759

September 16, 2008


Lecture outline

Lecture Outline

  • Background/discovery

  • siRNA/shRNA

  • Movie

  • miRNA

    • Biogenesis

    • Functions

    • Applications


Rnai and gene silencing

# Publications


Rnai and gene silencing

Focus on RNA interference - A user’s guide

September 2006


Rnai and gene silencing

1998 Feb 19;391(6669):806-11

No RNA

No probe

dsRNA

anti-sense ssRNA

In situ hybridization for mex-3 mRNA


Rnai and gene silencing

Nature 391:744 1998


Puzzles of the rnai

Puzzles of the RNAi

  • Both sense and anti-sense ssRNA effective

  • Catalytic – very few copies of dsRNA could silence abundant mRNA

  • Therefore not conventional antisense

  • Only dsRNA targeting mature mRNA are effective, not to introns or promoters

  • RNAi can cross cellular boundaries


Rnai and gene silencing

Fire, A, 2006 Nobel Prize Acceptance Speech


Rnai and gene silencing

Classes of small RNAs

dsRNA


Rnai dicing and slicing

RNAiDicing and slicing

  • All RNA silencing pathways are triggered by 21-27 nt long small RNAs

    • Small interfering RNAs – siRNA

    • Micro RNAs – miRNA

    • Piwi RNA

  • RNAi induction using long dsRNA only operates in plants and invertebrates

  • In mammals, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNaseL, degrades mRNA


Rnai and gene silencing

Fire, A, 2006 Nobel Prize Acceptance Speech


Pkr inhibition of translation

PKR Inhibition of Translation


Pkr inhibition of translation1

PKR Inhibition of Translation


Sirna and shrna

siRNA and shRNA

  • siRNA (short interfering RNA)

    • typically synthesized chemically then introduced into target cells

  • shRNA (short hairpin RNA)

    • typically introduced as a plasmid or viral vector

    • endogenous production, can be long term

    • enters the RNAi pathway upstream of siRNA


Rnai and gene silencing

Novina and Sharp Nature 430:161 2004


Dicer

Dicer

  • Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity


Rnai and gene silencing

RISC

  • RISC has helicase, endonucelase “slicer”,S and homology searching domains.

  • Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense strand.


Rnai and gene silencing

Fig. 1. Only mammalian Ago2 can form cleavage-competent RISC

Identification of Ago2

as “Slicer” in the RISC

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS


Rnai and gene silencing

Fig. 2. Argonaute2 is essential for mouse development

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS


Rnai and gene silencing

Fig. 3. Argonaute2 is essential for RNAi in MEFs

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS


Rnai and gene silencing

The ago1 mutant Arabidopsis develops abnormally because it does not produce an

effector of silencing. The Argonaute genes were so named because the mutant plants

look like an argonaute squid.

The Sainsbury LaboratoryJohn Innes CentreColney LaneNorwich, NR4 7UH, UK


Rnai and gene silencing

Summary of siRNA

and shRNA processing


Processing of sirna

Processing of siRNA

  • Which becomes guide strand in the RISC and which is excluded?

    • Sequence and structure

    • Strand with the less-tightly base pared 5’ end is incorporated becomes guide strand


Mirna

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 regulates separate genes and has imperfect complementarity. May be 100’s/miRNA. Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control

  • 30 – 50 % of genes regulated by miRNA


Mirna1

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.


Rnai and gene silencing

Tissue-specific

expression of miRNA

Plasterk RHA Cell 124:877 2006


Rnai and gene silencing

The structure of human pri-miRNAs

Du, T. et al. Development 2005;132:4645-4652


Overview of mirna biogenesis

Overview of miRNA biogenesis


Rnai and gene silencing

Cullen Nature Immunology 7:563 2006


Processing of mirna

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.


Rnai and gene silencing

Imperfect homology between miRNA and 3’ UTR of target mRNA

Seed sequence has perfect homology


Players in mirna biogenesis

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


Players in mirna and sirna

Players in miRNA and siRNA

  • Argonaute (AGO)

    • PAZ domain binds the characteristic two-base 3' overhangs of siRNAs

    • PIWI domain: dsRNA guided hydrolysis of ssRNA

    • Slicer in RISC

  • Dicer (DCR)

    • Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA

  • TRBP

    • Cofactor for Dicer

  • RISC

    • RNA induced silencing complex


Mechanism of mirna suppression of gene expression

Mechanism of miRNA suppression of gene expression

  • Transcription

  • mRNA degradation

  • Translational repression

    • 1 Initiation

    • 2 Post-initiation step

  • Co-translational degradation of the nascent peptide


Mechanism of mirna suppression of gene expression1

Mechanism of miRNA suppression of gene expression

  • Translational repression

    • 1 Initiation

    • 2 Post-initiation step

How to distinguish?


Mirna can repress and activate translation

miRNA can repress and activate translation


Mirna2

miRNA

  • miRNA in disease

    • Loss of function mutation of miRNA

    • Gain of function mutation of miRNA, e.g overexpression

    • Mutation of target site, no longer binds miRNA

    • Mutation of target site, now binds miRNA

    • Tumor suppressors

    • Oncogenes “oncomirs”


In vivo applications of rnai

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


Liver damage in mice expressing shrna long term

Liver damage in mice expressing shRNA long-term

Couzin Science 312:1121 2006

Grimm, et al. Nature 441:537-541 2006


Off target effects

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


Rnai and gene silencing

Toll-like receptors (TLR) can

recognize dsRNA


Rnai and gene silencing

Schizosaccharomyces pombe has DCR and AGO but not in Saccharomyces cerevisiae

Taphrina S. pombe S. cerevisiae Morel Penicillium

http://www.glocalbeer.dkhttp://tolweb.org/tree?group=Ascomycota&contgroup=Fungi

Swahili word for beer (Pombe)


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