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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




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

No RNA

No probe

dsRNA

anti-sense ssRNA

In situ hybridization for mex-3 mRNA



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 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





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


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


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.


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


Fig. 2. Argonaute2 is essential for mouse development

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

Published by AAAS


Fig. 3. Argonaute2 is essential for RNAi in MEFs

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

Published by AAAS


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


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.


Tissue-specific

expression of miRNA

Plasterk RHA Cell 124:877 2006


The structure of human pri-miRNAs

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



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.



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?



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



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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