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RNA Silencing RNAi. Post transcriptional gene regulation by siRNA and miRNA Pete Burrows MIC 759 October 26, 2006. Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA. Guillier, et al. Genes and Development 20:2338, 2006. Number of Publications.

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rna silencing rnai

RNA SilencingRNAi

Post transcriptional gene regulation by siRNA and miRNA

Pete Burrows

MIC 759

October 26, 2006


Small untranslated regulatory RNAs

Eukaryotes ncRNA

Prokaryotes sRNA


Number of Publications

Entrez PubMed Search Terms: RNAi or siRNA or miRNA

lecture outline
Lecture Outline
  • Discovery
  • General features
  • siRNA
    • Role of translation
  • miRNA
  • Problems

Focus on RNA interference - A user’s guide

September 2006

Nature Genetics

June 2006 Supplement


391:806, 1998


No probe


anti-sense ssRNA

In situ hybridization for mex-3 mRNA 4 cell embryos


News and Views

Nature 391:744 1998


In vitro transcription

Restriction digest

Blunt end

5’ overhang

3’ overhang

rnai dicing and slicing
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
  • Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity
  • Fly Dicer requires ATP, human may not
  • 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
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 an

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

look like an argonaute squid.

Knew that Ago a RISC component

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

identification of argonaute 2 as slicer in humans
Identification of Argonaute 2 as Slicer in humans

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

cytosolic players in sirna and mirna
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
schizosaccharomyces pombe has dcr and ago but not in s accharomyces cerevisiae

Taphrina S. pombe S. cerevisiae Morel Penicillium


Swahili word for beer (Pombe)

Schizosaccharomyces pombe has DCR and AGO but not in Saccharomyces cerevisiae

Ferritin IRE-IRP

Shen, et al. Differentiation 73:287-293 2005






Fe + - + - + - + -


7:633, 2005

7:719, 2005


RCK human homolog of yeast Dhh 1p

Interacts with eIF4e and represses translation

Targets RNA to P-bodies?

Sen and Blau, The FASEB Journal, 2006 20:1293



The Movie

Nature Genetics


  • 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.
  • 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
  • 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.

The structure of human pri-miRNAs

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

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

The miRNA biogenesis pathway

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

mechanism of mirna suppression of gene expression
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

Polyribosome association of lin-14 mRNA is unchanged

between L1 and L2

Polysome Profiles

lin-14 RNase protection assay


Polysome Profiles

lin-14 RNase protection assay

EDTA sensitivity of polysomes


Fig. 1. miRNAs target the initiation step of translation

No eIFs or Met-tRNAimet

Humphreys, David T. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 16961-16966

Copyright ©2005 by the National Academy of Sciences


Scanning model of Cap-dependent Translation Initiation





Fátima Gebauer & Matthias W. Hentze

Nature Reviews Molecular Cell Biology

5, 827-835 (2004)




other studies
Other Studies
  • Both 5’ cap and 3’ poly(A) tail are necessary but not sufficient for miRNA repression of translation
    • 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
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?
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
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
  • Recently found in flies