RNA interference

• In 1998, Andrew Fire and colleagues, demonstrated that RNAi is

a post-transcriptional process that involves mRNA degradation.
• The presence of short pieces of dsRNA called short interfering
RNA (siRNA) in cells undergoing RNAi.
• First observed in the roundworm C. elegans, RNAi refers to the
ability of double-stranded RNA to block expression of its
corresponding single - stranded mRNA.
• This phenomenon was called by several names: Cosuppression
and Post-transcriptional gene silencing (PTGS) in plants, RNA
interference (RNAi) in animals such as nematodes
(Caenorhabditis elegans) and fruit flies, and Quelling in fungi.
• The phenomenon of RNAi rests on the general ability of eukaryotic
cells to cleave double stranded RNA into short (23-nt) double-
stranded segments known as short interfering RNA (siRNA).
• The RNA endonuclease that catalyzes this reaction, known as
Dicer, is found in all metazoans.
• The siRNA molecules, in turn, can cause cleavage of mRNA
molecules of matching sequence, in a reaction catalyzed by a protein
complex known as RISC (RNA-induced silencing complex).

• RISC mediates recognition and hybridization between one strand of

the siRNA and its complementary sequence on the target mRNA;
subsequently, specific nucleases in the RISC complex cleave the
mRNA-siRNA hybrid.
• The normal function of both Dicer and RISC is to allow for gene
regulation by small endogenous RNA molecules known as micro-
RNAs, or miRNAs.
• RNA interference (RNAi) was discovered unexpectedly during attempts to
experimentally manipulate the expression of specific genes.

• Initially a long double-stranded RNA that mediates interference is processed

into a double stranded short interfering RNA (siRNA).

• The strands in siRNA contain 21-23 nucleotides hybridized to each other so that
the two bases at the 3' end of each strand are single stranded.

• The cytoplasmic double stranded RNA-specific ribonuclease that cleaves long

double-stranded RNA into siRNAs is the Dicer enzyme which is also involved
in processing pre-miRNAs.

• In both cases, the mature short single-stranded RNA, either mature siRNA or
mature miRNA, is assembled into RISC complexes in which the short RNAs
are bound by an Argonaute protein.
• What distinguishes a RISC complex containing an siRNA from one
containing an miRNA is that the siRNA base-pairs extensively with
its target RNA and induces its cleavage,
• whereas a RISC complex associated with an miRNA recognizes its
target through imperfect base-pairing and results in inhibition of
translation.
• The Argonaute protein is responsible for cleavage of target RNA;
one domain of the Argonaute protein is homologous to RNase H
enzymes that degrade the RNA of an RNA-DNA hybrid.
• When the 5' end of the short RNA of a RISC complex base-pairs
precisely with a target mRNA over a distance of one turn of an RNA
helix (10-12 base pairs), this domain of Argonaute cleaves the
phosphodiesrer bond of the target RNA across from nucleotides 10
and 11 of the siRNA.
• The cleaved RNAs are released and subsequently degraded by
cytoplasmic exosomes and 5' exoribonucleases.
• This process of RNA interference is believed to be an ancient
cellular defense against certain viruses and mobile genetic
elements in both plants and animals.
• Most miRNAs are processed from RNA polymerase II
transcripts of several hundred to thousands of nucleotides in
length called pri (for primary transcript)-miRNAs
• PrimiRNAs can contain the sequence of one or more miRNAs.
• Within these long transcripts are sequences that fold into
hairpin structures of ~70 nucleotides in length with imperfect
base pairing in the stem.
• A nuclear RNase specific for double-stranded RNA called
Drosha acts with a nuclear double-stranded RNA binding
protein called DGCR8 in humans (Pasha in Drosophila) and
cleaves the hairpin region out of the long precursor RNA,
generating a pre-miRNA.
• Pre-miRNAs are recognized and bound by a specific
nuclear transporter, Exportin5, allowing the complex to
diffuse through the inner channel of the nuclear pore
complex.
• Once in the cytoplasm, a cytoplasmic double-stranded
RNA-specific RNase called Dicer acts with a cytoplasmic
double-stranded RNA-binding protein called TRBP in
humans (Tar binding protein) to further process the pre-
miRNA into a double stranded miRNA.
• Finally, one of the two strands is selected for assembly into
a mature RNA-induced silencing complex (RISC)
containing a single-stranded mature miRNA bound by a
multidomain Argonaute protein, a member of a protein
family with a recognizable conserved sequence.
• The miRNA-RISC complexes associate with target mRNPs by
base pairing between the Argonaute-bound mature miRNA and
complementary regions in the 3' untranslated regions (3'
UTRs) of target mRNAs.
• Inhibition of target mRNA translation requires the binding of
two or more RISC complexes to distinct complementary
regions in the target mRNA 3' UTR.
• Binding of RISC complexes causes the bound mRNPs to
associate with dense cytoplasmic domains called cytoplasmic
RNA-processing bodies, or simply P bodies.
• P bodies, are sites of RNA degradation that contain no
ribosomes or translation factors, potentially explaining the
inhibition of translation.
Difference between miRNA and siRNA
• Function of both species is regulation of gene expression.
• Difference is in where they originate.
• siRNA originates with dsRNA.
• siRNA is most commonly a response to foreign RNA (usually
viral) and is often 100% complementary to the target.
• miRNA originates with ssRNA that forms a hairpin secondary
structure.
• miRNA regulates post-transcriptional gene expression and is
often not 100% complementary to the target.
• Researchers exploit the micro-RNA pathway for intentional
silencing of a gene of interest by using either of two general
methods for generating siRNAs of defined sequence.
• In the first method, a double-stranded RNA corresponding to the
target-gene sequence is produced by in vitro transcription of
both sense and antisense copies of this sequence.
• This dsRNA is injected into the gonad of an adult worm, where
it is converted to siRNA by Dicer in the developing embryos.
• In conjunction with the RISC complex, the siRNA molecules
cause the corresponding mRNA molecules to be destroyed
rapidly.
• The resulting worms display a phenotype similar to the one that
would result from disruption of the corresponding gene itself.
• The second method is to produce a specific double stranded
RNA in vivo.
• An efficient way to do this is to express a synthetic gene that is
designed to contain tandem segments of both sense and
antisense sequences corresponding to the target gene.
• When this gene is transcribed, a double-stranded RNA
"hairpin" structure forms, known as small hairpin RNA, or
shRNA.
• The shRNA will then be cleaved by Dicer to form siRNA
molecules.
• The lentiviral expression vectors are particularly useful for
introducing synthetic genes for the expression of shRNA
constructs into animal cells.
• Both RNAi methods lend themselves to systematic studies to
inactivate each of the known genes in an organism and to
observe what goes wrong.
• Other organisms in which RNAi-mediated gene inactivation
has been successful include Drosophila, many kinds of plants,
zebra fish, the frog Xenopus, and mice.