RNA silencing/RNAi involves the knock-down of genes through two types of small RNA molecules, miRNAs and siRNAs, that are involved in post-transcriptional and transcriptional gene silencing as an antiviral mechanism; short double-stranded RNAs are processed by the Dicer enzyme into siRNAs which are incorporated into the RISC complex to guide degradation of homologous mRNA targets; RNAi is an important endogenous gene regulatory mechanism and has applications in gene function analysis, gene therapy, and cancer treatment.

1. RNA Silencing/RNAi

2. RNA SILENCING
• The process of knock-down of any gene
• Functions as a antiviral mechanism
• May be post transcriptional as well as transcriptional gene
silencing
• Two types of short RNA molecules are involved
 miRNA
 siRNA

3. Short history of post-transcriptional gene silencing
1990 Jorgensen :
Introduction of transgenes homologous to
endogenous genes often resulted in plants with both
genes suppressed!
Called Co-suppression
Resulted in degradation of the endogenous and the
transgene mRNA

4.  Discovery
Inhibition by injected
anti-sense RNA
Sense RNA had similar Jorgensen et al., Nature (1988)
effects!
Napoli, Lemieux, Jorgensen. Plant Cell (1990)

5. Amin, I and K. Hussain 2011

6. Contd….
1995 Guo and Kemphues:
-injection of either antisense or sense RNAs in the
germline of C. elegans was equally effective at
silencing homologous target genes
1998 Mello and Fire:
-extension of above experiments, combination of
sense and antisense RNA (= dsRNA) was 10
times more effective than single strand RNA

7. Source for siRNA:
Endogenous
Exogenous

8. double-stranded RNAs are produced by:
– transcription of inverted repeats
– viral replication
– transcription of RNA by RNA-dependent RNA-
polymerases (RdRP)
double-stranded RNA triggers cleavage of
homologous mRNA
PTGS-defective plants are more sensitive to infection
by RNA viruses
in RNAi defective nematodes, transposons are much
more active

9. Dicer
Double-stranded RNA triggers processed into siRNAs
by enzyme RNAseIII family, specifically the Dicer family
Dicer family proteins are ATP-dependent nucleases.
These proteins dice long dsRNA into small RNA
duplexes of 21-26nt sizes.
Loss of dicer: loss of silencing process

10. Citwood and Timmermans, Nature 2010

11. 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

13. Spreading of silencing and amplification
via RdRP (RNA-dependent RNA polymerase)
S. pombe, plants, worms
systemic silencing; amplification of silencing
Not in Drosophila or in humans

14. miRNA siRNA
noncoding RNA gene repeat associated
PolII transcribed PolII/V transcribed or
bidirectional transcription
Hairpin (loop, mismatches) double stranded RNA
PTGS PTGS
TGS? TGS

15. Biogenesis of microRNA
Cullen Nature Immunology 7:563 2006

16. Translational Repression

17.  Functions --- RNA-Directed De novo Methylation (RdDM)
DCL3, 24 nt siRNAs, AGO4, MET1/DRM2
AGO4
Marjori A. Matzke & James A. Birchler
Nature Reviews Genetics 6, 24-35 (2005)

18. Why is RNAi important?
begun in worms, flies, and plants - as an accidental
observation.
general applications in mammalian cells.
probably much more common than appreciated
before:
– it was recently discovered that small RNAs
correspond to centromer heterochromatin repeats
– RNAi regulates heterochromatic silencing
Faster identification of gene function

19. Most widely held view is that RNAi evolved to
protect the genome from viruses (or other
invading DNAs or RNAs)
Recently, very small (micro) RNAs have been
discovered in several eukaryotes that regulate
developmentally other large RNAs
– May be a new use for the RNAi mechanism
besides defense

20. Contd…..
Powerful for analyzing unknown genes in
sequence genomes.
 efforts are being undertaken to target every
human gene via miRNAs
Gene therapy: down-regulation of certain
genes/mutated alleles
Cancer treatments
– knock-out of genes required for cell proliferation
– knock-out of genes encoding key structural
proteins