This document discusses RNA interference (RNAi), a biological mechanism that leads to post-transcriptional gene silencing triggered by double-stranded RNA molecules. It was discovered in 1998 by Fire and Mello, who received the Nobel Prize for this work. The mechanism involves double-stranded RNA being processed by the enzyme Dicer into small interfering RNAs that integrate into the RNA-induced silencing complex and guide mRNA degradation. RNAi can be induced by synthetic siRNAs or endogenous microRNAs and represents a powerful tool for studying gene function and developing therapies.

1. Doctoralseminar
SK Singh,
Advisor
Associate Professor Cum Senior
Scientist
Ankita SinhaPhD Student
Dr Rajendra Prasad
Central Agricultural University, Pusa, Samastipur, Bihar
RNA interference

2. RNA interference (RNAi) is a biological mechanism which leads to post
transcriptional gene silencing (PTGS) triggered by double stranded RNA (dsRNA)
molecules to prevent the expression of specific genes
Introduction
It’s basically an RNA-dependent gene silencing process
Here mRNA is unable to make a protein during translation.

3. Repression of transcription mRNA degradation
transcriptional gene silencing
(TGS)
Post transcriptional gene
silencing (PTGS)
The mechanism of RNA interference is discovered
by Andrew Fire and Craig Mello in 1998, for which
they got Nobel prize in 2006.

4. The RNA causing gene silencing
may be endogenous or exogenous
Endogenous RNA that can silence
genes is known as micro RNA
(miRNA)
Endogenous RNA that can silence
genes is known as micro RNA
(miRNA)
siRNA can be of viral origin or synthetic

5. Gene silencing phenomenon were also observed independently in fungal system,
called as Quelling found in Neurospora crassa
Romano and Macino, 1992
Gene silencing could be used to treat persistent infections by turning off antibiotic
resistance genes in bacteria
Gene silencing by a pathway called RNA interference has proved a powerful tool for
biologists, who have used the technique to silence particular genes in order to study
their functions in disease development.

6. • This experiment was first done by Zamecnik
and Stephenson in 1978 and continues to be a
useful approach, both for laboratory
experiments and potentially for clinical
applications (antisense therapy).

7. Entrance of a double-stranded RNA (dsRNA) Into the cell.
dsRNA is processed by Dicer into short RNA duplexes.In most
species, cleavage of longer double-stranded RNAs by Dicer
produces double-stranded siRNAs and miRNAs that are
approximately ∼21 nucleotides long.
These small RNAs subsequently associate in a RNA induced
silencing complex (RISC.) which contain ARGONAUTE (AGO)
proteins
Argonaute catalyses cleavage near the centre of the region of
the mRNA that is bound by the siRNA.
After the mRNA has been cleaved by RISC exonucleases can
degrade it
RNA interference pathway

8. • miRNAs were discovered by Ambros et al (1993)
in a nematode, C. elegans
• ds RNA causing RNA interference were
discovered by Fire and Mello (1998) in C. elegans
• The dsRNA (double-stranded RNA) came to be
known later as siRNA

9. siRNA
it is used to silence desired
genes
it’s a synthetic RNA duplex
designed to specifically
target a particular mRNA
for degradation.
The 3’ end of each strand overhangs the 5’ end of the other strand
The overhang is two nucleotides long

14. • siRNA integrates into a protein complex in an ATP-
dependent reaction
• siRNA direct RISC to bind to specific mRNA, the
targeting is precised because it determined by base
pairing between siRNA and the mRNA.
• siRNA often have perfect complementarty to
target sites,once bound the argonaute catalyzes the
cleavage of mRNA which will then be degraded.

15. Mi RNA (micro RNA):
• Originate from capped & polyadenylated full length
precursors (primiRNA)
• Hairpin precursor ~70 nt (pre-mi RNA)
• Mature mi RNA ~22 nt (mi RNA)
• mi RNA originates with SS RNA that forms a hairpin
secondary structure.
• Mi RNA regulates post-transcriptional gene expression
and is often not 100% complementary to the target.
• mi RNA help to regulate gene expression, particularly
during induction of heterochromatin formation serves
to down regulate genes pre- transcriptionally (RNA
induced transcriptional silencing or RITS)

17. RISC – The Catalytic Engine of RNAi
RISC mediates endonucleolytic cleavage of the target RNA, not its complete destruction.
RNAi effector complex
Critical for target mRNA degradation or translation inhibition
Not well characterized: 4 subunits? More?
Components:
(i) PAZ
(ii) PIWI
(iii) VIG (vasa intronic gene product)
(iv) Fragile X-related protein
(v) Nuclease Tudor-SN
Activities associated with RISC
– Helicase
– Endonuclease (Slicer) and exonuclease (or is it Dicer?)
– “homology seeking”/RNA binding
Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003]

18. • RISC is a large (~500-kDa) RNA-multi protein complex,
which triggers mRNA degradation in response to Si RNA
• Unwinding of double- stranded Si RNA by ATP independent
helicase.
• The active components of an RISC are endonucleases called
argonaute proteins which cleave the target mRNA strand.
RNA Inducing Silencing Complex (RISC)

19. DICER
• Cleaves dsRNA or pre-miRNA
• Leaves 3’ overhangs and 5’ phosphate groups
• Cytoplasmic RNAse-III enzyme
• Functional domains in Dicer [Bernstein et al., 2001]
(i) Putative helicase
(ii) PAZ domain (110-130 amino-acid domain present in protein like Argo,
Piwi..);it is thought to be important for protein-protein interaction
(iii) Tandem RNAse-III(RIIIa & RIIIb) domains
(iv) dsRNA binding domain in the carboxyl terminal segment
(v) DUF283 domain
• Multiple Dicer genes in Drosophila and plants. [He and Hannon, 2004]
• How does it identify pre-miRNA?
PAZ domain interact with dsRNA that present 2-nt 3’overhangs (result of
Drosha cut)
in plants
• – DCL1, a Dicer-like protein does its job.

20. • DICER’s domain:
1) Amino-terminal helicase domain
2) Dual RNAse III motifs
3) dsRNA binding domain
4) PAZ domain

22. • AGRONAUTE
• A protein complex (~130kDa) use to recognize the target mRNA
• Evolutionary conserved proteins represented in nearly all eukaryotic lineages.
• Ago protein sequence are defined by the presence of two conserved region of
homology.
• Consistently co-purifies with RISC
• Contains two conserved domains
PAZ
PIWI
“Homology seeking” activity?
• – Binds siRNA and miRNA 2004]
• – Distinguishes antisense strand and Sharp, 2004]
• Multiple Ago family proteins
• – Different RISCs?
• – Tissue specific? Developmentally regulated?
• Evidence for different et al., 2004]
• [Hammond et al.,2001]
• [Ekwall, 2004]
• [Novina and Sharp, 2004]
• RISCs. [Tijsterman et al., 2004]

23. Argonaute proteins contain PAZ and
Piwi domains
PAZ domain (Piwi-argonaute-zwille) :
RNA binding domain that recognizes
miRNA duplex ends.
Piwi : interaction with Dicer.

24. MECHANISM
• The process to silence genes first begins with the
entrance of a double-stranded RNA (dsRNA) molecule
into the cell,which triggers the RNAi pathway.
• The double-stranded molecule is then cut into small
double stranded fragments by an enzyme called Dicer.
• These small fragments, which include small interfering
RNAs (siRNA) and microRNA (miRNA), are
approximately 21–23 nucleotides in length.
• The fragments integrate into a multi-subunit protein
called the RNA-induced silencing complex, which
contains Argonaute proteins that are essential
components of the RNAi pathway.

25. • 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
• One strand of the molecule, called the "guide" strand, binds to
RISC, while the other strand, known as the "passenger" strand is
degraded.
• The guide or antisense strand of the fragment that remains bound
to RISC directs the sequence-specific silencing of the target mRNA
molecule.
• The genes can be silenced by siRNA molecules that cause the
endonucleatic cleavage of the target mRNA molecules or by miRNA
molecules that suppress translation of the mRNA molecule.
• RNAi is thought to have evolved as a cellular defense mechanism
against invaders, such as RNA viruses, or to combat the proliferation
of transposons within a cell's DNA. Both RNA viruses and
transposons can exist as double-stranded RNA and lead to the
activation of RNAi.

27. Role of RNAi in plant system
• Production of Banana varieties resistant to the Banana
Bract Mosaic Virus (BBrMV) by RNAi
(Rodoni et al. )
Applications of RNAi
RNAi technology can be used to identify and
functionally assess the thousands of genes within the
genome that potentially participate in disease
phenotypes. In addition, RNAi technology provides an
efficient means for blocking expression of a specific
gene and evaluating its response to chemical
compounds or changes in signaling pathways.

28. • When dsRNA molecules produced during viral
replication trigger gene silencing, the process
is called virus-induced gene silencing (VGS).
• One interesting feature of RNA silencing in
plants is that once it is triggered in a certain
cell, a mobile signal is produced and spread
through the whole plant causing the entire
plant to be silenced.
• Cladosporium fulvum Magnaporthae oryzae,
Venturia inaequalis and Neurospora crassa

29. • Advantages of gene silencing:
• Down regulation of gene expression simplifies "knockout“ analysis.
• Easier than use of antisense oligonucleotides. Si RNA more effective
and sensitive at lower concentration.
• Cost effective
• High Specificity middle region 9-14 are most sensitive With SiRNA,
the researcher can simultaneously perform experiments in any cell
type of interest Can be labelled Ease of transfection by use of
vector
• blocking expression of unwanted genes and undesirable substances.
• Inducing viral resistance
• Powerful tool for analysing unknown genes in sequenced genomes.
• Useful approach in future gene therapy.
• Oligonucleotides can be manufactured quickly, some within one
week; the sequence of the mRNA is all that is needed

30. • How RNA interference (RNAi) works
RNAi technology takes advantage of the cell’s
natural machinery, facilitated by short interfering
RNA molecules, to effectively knock down
expression of a gene of interest. There are
several ways to induce RNAi, synthetic molecules,
RNAi vectors, and in vitro dicing (Figure 2). In
mammalian cells, short pieces of dsRNA, short
interfering RNA (siRNA), initiate the specific
degradation of a targeted cellular mRNA.