2. Gene expression can be silenced by
some small RNA molecules
This phenomenon has been termed
as RNA interference (RNAi)
It was first seen in plants but later found
in mammals including human beings
3. RNA interference is an RNA-dependent
gene silencing process
The RNA causing gene silencing may be
endogenous or exogenous
4. Endogenous RNA that can silence genes
is known as micro RNA (miRNA)
Exogenous RNA that can silence genes is
known as small interfering RNA (siRNA)
siRNA can be of viral origin or synthetic
5. miRNAs were discovered by Ambros et al
(1993) in a nematode, C. elegans
Fire and Mello (1998) discovered ds RNA
causing RNA interference in C. elegans
The dsRNA (double-stranded RNA) came
to be known later as siRNA
6. Both siRNA and miRNA are formed from
large precursors
The precursor is cleaved to form siRNA or
miRNA
7. The precursor of siRNA is a stem-loop
structure
This is cleaved to form double-stranded
siRNA
Cleavage is catalysed by Dicer, an
enzyme of ribonuclease III family
siRNA
8. siRNA is 21-23 nucleotides in length
The 3β end of each strand overhangs the
5β end of the other strand
The overhang is two nucleotides long
OH
HO
β
β3β
3β5β
5β
9. siRNA is integrated into a protein complex
in an ATP-dependent reaction
The complex is termed as RNA-induced
silencing complex (RISC)
The active components of RISC are some
endonucleases called argonaute proteins
10. Out of the two strands of siRNA, only
one directs gene silencing
This strand is known as the guide strand
The other strand is known as passenger
(anti-guide) strand
11. The passenger strand is degraded by an
argonaute protein
This results in activation of RISC
The guide strand finds and binds to a
complementary mRNA
After this binding, argonaute proteins
cleave the mRNA
13. Synthetic siRNAs can be used to silence
desired genes
The siRNA should have a sequence
complementary to mRNA of the target gene
14. siRNAs as drugs
siRNAs have been tried as drugs to
silence specific genes in human beings
Some success has been reported in:
Age-related macular degeneration
Cancer
15. Several problems are yet to be solved in
therapeutic use of siRNAs
Immune system can destroy the siRNA
mistaking it as viral RNA
Homology in base sequence of genes can
result in silencing of unintended genes
Problems with siRNAs as drugs
16. Response of different types of cells to
siRNAs is not uniform
Some cells respond very well whereas
others show a poor response
17. A mature miRNA is 20-25 nucleotides
long
It is formed from a much longer
precursor
The primary transcript is known as
primary miRNA (pri-miRNA)
miRNA
18. pri-miRNA is processed in the nucleus
It is converted into a 70-nucleotide long
stem-loop structure
The stem-loop structure is called pre-
miRNA
pre-miRNA
19. The pre-miRNA goes to cytoplasm
The double stranded portion of pre-miRNA
is cleaved by Dicer
This produces mature miRNA molecule
One to six miRNAs can be formed from a
single pre-miRNA precursor
20. miRNA is integrated into RNA-induced
silencing complex (RISC)
From this point, siRNA and miRNA share
the same downstream pathway
21. The guide strand binds to a comple-
mentary mRNA molecule
The bound mRNA is either degraded or
silenced
If the base-complementarity is perfect,
the mRNA is degraded by the argonautes
23. More commonly, base-complementarity
between miRNA and mRNA is imperfect
Even if the complementarity is imperfect,
miRNA is partially bound to mRNA
mRNA partially bound to miRNA cannot
be translated
Therefore, gene expression is effectively
silenced
24. Generally pairs
with the target
imperfectly
Can inhibit several
targets having
similar sequences
miRNAsiRNA
Pairs with
the target
perfectly
Induces mRNA
cleavage of a
specific target
Differences between siRNA and miRNA
25. The miRNAs are formed from trans-
cription of sequences located:
Either in between genes
Or in introns
Human beings may synthesize more
than 1,000 miRNAs
Nearly 40% of the miRNAs are formed
from introns
26. The miRNAs may target about 60% of
genes
Natural function of miRNAs may be in
the regulation of gene expression
27. Different sets of miRNAs are expressed
in different cell types and tissues
The miRNAs may regulate:
Tissue-specific expression of
genes
Temporal expression patterns
seen during development