This video speaks about RNA interference

1. POST TRANSCRIPTIONAL GENE
SILENCING (PTGS)
DR ARPITA MISHRA

2. Gene silencing
Interruption or suppression of the expression of a gene at transcriptional or translational levels.
Two classes:
• Transcriptional Gene silencing
• Post Transcriptional gene silencing
Post-transcriptional gene silencing (PTGS) is a mechanism that degrades specific messenger RNAs and thereby
reduces the expression of a specific gene.
PTGS has many names:
• Co-suppression in plants,
• Quelling in fungi
• and RNA interference (RNAi) in animals,
In all cases, mRNA is degraded to decrease gene expression.
The process is homology dependant
PTGS mechanisms naturally occur in organisms

3. PTGS can be artificially induced by the introduction of double-stranded RNA (dsRNA)
• A potent tool in functional genomics
• Is used as a tool to knock down expression of specific genes in a variety of organisms
• Therapeutic applications in cancer, infectious diseases and neurodegenerative diseases
History
Co-suppression was observed for the first time in petunias.
In a Genetic Engineering experiment, Rich Jorgensen and colleagues in1990 introduced pigment-producing genes
under the control of a powerful promoter into petunia plants
The genes introduced were chalcone synthase ChsA transgene or a dihydroflavonol-4-reductase transgene.Both
genes encoded proteins involved in the production of anthocyanin pigments.
The intention was to deepen the purple colour of petunia flowers. However many flowers appeared variegated
or even white
Jorgensen named the observed phenomenon "cosuppression", since the expression of both the introduced gene
and the homologous endogenous gene was suppressed

5. RNA –I
Fire and Mello first injected dsRNA — a mixture of both sense and antisense strands — into C. elegans.
The investigators injected dsRNA corresponding to a 742-nucleotide segment of unc22 gene into either the
gonad or body cavity region of an adult nematode
unc22 encodes an abundant but nonessential myofilament protein, and the decrease in unc22 activity is
supposed to produce an increasingly severe twitching phenotype
The injected animal showed weak twitching, whereas the progeny individuals were strong twitchers.
Thus it was shown that exogenous introduction of dsRNA corresponding to a gene could result in silencing of
the endogenous gene
The injection resulted in much more efficient silencing than injection of either the sense or the antisense strands
alone. Indeed, injection of just a few molecules of dsRNA per cell was sufficient to completely silence the
homologous gene's expression.
Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm,
but also in its first generation offspring
This process, of PTGS by the introduction of dS RNA was named RNA interference

6. FEATURES OF RNA-I
• Features of the RNA-i mechanism are:
• The process is initiated by the dsRNA,
• The target RNA is degraded in a homology dependent fashion, and
• The degradative machinery requires a set of proteins which are similar in structure and function across
most organisms
In most of these processes, certain invariant features are observed, including
• the formation of small non coding RNAs like small interfering RNA (siRNA), microRNA (miRNA) from
the dsRNA that initiates the process
• The systemic transmission and amplification of the silencing signal from its site of initiation.

7. si RNA AND mi RNA
• si RNA, mi RNA are non-coding RNAs that are associated with RNA interference
• si RNA (or short/small interfering RNA) are produced either from exogenous (provided from the
outside) ds RNA introduced into the cell in the form of synthetic constructs/viral DNA. It can also be
generated endogenously
• mi RNA (micro RNA) are always produced from endogenous gene products (coded by the genome)
• As much as 5% of the human genome is dedicated to encoding and producing the >1,000 miRNAs that
regulate at least 30% of our genes
•

8. DICER
• Dicer is the enzyme that cleaves dsRNA into siRNA
• It belongs to the rnase III nuclease family
• Rnase III family members are among the few nucleases that show specificity for dsRNAs
• Dicer has four distinct domains:
• An amino-terminal helicase domain,
• Dual RNAse III motifs,
• A dsRNA binding domain, and
• A PAZ domain (a 110-amino-acid domain present in proteins like piwi, argo, and zwille/pinhead)
• Cleavage by dicer is thought to be catalyzed by its tandem rnase III domains.

9. RNA INDUCED SILENCING COMPLEX(RISC)
• Is a multicomponent protein complex
• One of the protein components of this complex was identified as a member of the argonaute family of
proteins and was termed argonaute2 (AGO2)
• AGO2 is a 130-kda protein containing polyglutamine residues, PAZ, and PIWI domains characteristic of
members of the argonaute gene family
• AGO2 bears catalytic activity and can cleave the target mRNA
• RISC also contains other proteins including RNA binding proteins, helicase proteins etc
• A few molecules of dsRNA are sufficient to degrade a continuously transcribed target mRNA for a
long period of time.

10. siRNA MEDIATED RNA-I
Initiation:
• processing of dsRNA into siRNAs
• Input dsRNA introduced directly or via a transgene or virus is digested into 21-23 nucleotide small
interfering RNAs (siRNAs)
• siRNAs are formed and accumulate as double-stranded RNA molecules of defined chemical
structures
• Each strand of siRNA has 5-phosphate and 3-hydroxyl termini and 2- to 3-nucleotide 3 overhangs.
• SiRNAs are produced when the enzyme dicer, a member of the RNase iii family of dsRNA-specific
ribonucleases, cleaves dsRNA in an atp-dependent manner

11. Effector step:
• The si RNA is then loaded on to a protein complex called as RNA induced silencing complex(RISC).
• The loading is assisted by the risc-loading complex (RLC) comprised of the DICER and dsRNA-binding
protein
• Inside the RISC the siRNA unwinds in an ATP dependant activity.
• One of the strands is called the guide strand (the strand complementary to the cognate mRNA) and the
other is called the passenger strand.
• The passenger strand is discarded
• The guide strand remains associated with the argonaut protein of the RISC
• The guide strand targets the RISC to the cognate mRNA and the RISC proteins through diverse
mechanisms prevent the mRNA from being translated