RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is a conserved biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes. Small pieces of RNA can shut down protein translation by binding to the messenger RNAs that code for those proteins. RNA interference is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes.

1. RNAi and its
importance

2. Protein
synthesis
Ribosome
mRNA mRNA
antisensemRNA
Ribosome
can’t bind
X
Noprotein
synthesis
AntisenseRNAinhibitsprotein synthesis by blocking Translation

3. What is RNAi?
• RNA interference (RNAi)
• Cellular process by which an mRNA is targeted for
degradation by ds RNA with a strand complementary
to a fragment of such mRNA.
• Responsible for post-transcriptional gene silencing of
the gene from which it was derived
• Specific terms for gene silencing:-
• Post transcriptional gene silencing (PTGS) – Plants
• Quelling – Fungi
• RNA interference(RNAi) – Animals

4. • RNA phenomenon was discovered in transgenic plants
Petunia hybrida L. (Napoli et al. 1990)
• They wanted to enhance anthocyanin pigments.
• Unexpectedly, transgenic plants producing white or
chimeric flowers were obtained instead of dark purple
flowers due to the silencing of endogenous homologous
gene and this phenomenon was termed as Co-
suppression.

5. Co-supression
of genes
(PTGS)
TIME LINE
1990 1992 1998 2001 2003 2004
2002 2005 2006
Quelling in
Neurospora
RNAi in
C. elegans
siRNA in
Mamalian cells
Scienists named RNAi
As “ Technology of
the year”
RNAi mechanism
discovered
Nobel Prize Fire and
Mello

6. Nobel Prize in Physiology and medicine
2006
• Andrew Fire has been professor of pathology and genetics
at Stanford university School of medicine, Stanford,
California, USA.
• He did his PhD in biology at the MIT, USA. When Fire and
Mello made their key discoveries about RNA interference,
Fire was working at the Carnegie institution of
Washington.
• Craig Mello is a professor of Molecular medicine.
Since 1994 he has worked within the program in
Molecular Medicine, University of Massachusetts
Medical Scholl, Worcester, USA.
• He too his PhD in cellular and developmental
Biology at Harvard University.

8. Dr. Ramanjulu Sunkar

10. Multiple pathways for RNAi
There are two main RNAi pathway:
• small interfering RNAs (siRNAs) generated via
processing of longer dsRNA.
• micro RNAs (miRNAs) that are generated via
processing of stem loop precursors
(transcribed in the nucleus).

11.  21-23 nucleotide dsRNA that mediatePTGS
 Produced in vivo by cleavage of dsRNA
 Amplification by an RNA-dependent RNA polymerase (RdRP)
may occur
 Incorporated into the RISC guiding it to mRNA
 Complementary to a specific sequence of target mRNA for
degradation.

12.  single-stranded RNAs of 22-nt that are processed from ~70-nt
hairpin RNA precursors by Rnase III nuclease Dicer
 Similar to siRNAs
 can silence gene activity via destruction of homologous
mRNA in plants or blocking its translation in plants and
animals.

13. Main Component of RNAi machinery
for siRNA generation
There are four components for siRNAs
generation:
• DICER
• small interfering RNA (siRNA)
• RNA-induced silencing complex (RISC)
• RNA-dependent RNA polymerase (RdRP)

14. DICER
• Discovered by Berbstein et al. (2001) in drosophilla
• Enzyme belongs to the RNase III ribonuclease family
• initiates RNAi
• Cutting mechanism of DICER enzyme is ATP-dependent
(Kuznetsov 2003)
• Responsible for processing of dsRNA into siRNA containing 21-
24 nt
• cleaves dsRNA and leaves 3' overhangs and 5' phosphate
groups.

15. Argonaut (AGO)
• Consistently co-purifies with RISC. (Hammond et.al, 2001)
• Binds with siRNA and miRNA and distinguishes antisense
strand (Novina and sharp 2004)
• Homology seeking activity.

16. RNA induced silencing complex (RISC)
• discovered in drosophilla (hammond et. al (2000))
• consists of both protein and RNA
• targets and destroy endogenous mRNA complimentary to
siRNA.
• activities associated with RISC:-
i. helicase
ii. RNA binding
iii. endonuclease and exonuclease slicer

17. RNA dependent RNA polymerase
(RdRP)
• Triggeringandamplifying the silencingeffect
• Systemicnature of RNAi
• Activity found in plants andC.elegans
• MayexplainefficiencyofRNAi
• Requiredfor RNAi?
a. Not found in mammalsordrosophila
b. RdRPdeficient plants and worms... Resultsnotdecisive

18. MECHANISM/PROCESSES
 Induction phase
 Completion phase
 Multiplication phase
 Degradation phase

19. Induction Phase
Completion
Phase
Multiplication
Phase
Degradation
Phase
dsRNA
RISC RISC
mRNA
mRNA
degradation
siRNA
DICER
small RNAs as
guiding signals
ATP mediated
unwinding

20. DICER
dsRNA cut by DICER (endonuclease protein)
dsRNA
siRNA
siRNA
siRNA
siRNA
3' overhang

21. RISC complex formation and unwinding
Argonaute(
AGO)
RISC complex
siRNA
ATP mediated
unwinding
+
antisense siRNA protein
complex/ guide strand
sense RNA

22. +
RISC
mRNA
binding of RISC with
mRNA
Argonaut catalyzes cleavage
of mRNA
Degradation

23. Jagtap et.al., 2011

24. MaincomponentofRNAi machineryfor miRNA
generation
ThereareFIVEcomponent for miRNAsgeneration
1. Drosha
2. Dicer
3. Argonaut(Ago)
4. RNA-InducedSilencingComplex(RISC)
5. RNA-DependentRNAPolymerase(RdRP)

25.  Processespri-miRNAinto pre-miRNA
– Leaves3’ overhangs onpre-miRNA
 NuclearRNAse-IIIenzyme[Leeat al.,2003]
– TandemRNAse-IIIdomains
 Howdoesit identifypri-miRNA?
– Hairpin terminal loopsize
– Stemstructure
– Hairpin flankingsequences
 Not yet found inplants
– Maybe Dicer doesitsjob?

26. miRNA biogenesis
 Transcribedfrom endogenousgeneaspri-miRNA
– Primary miRNA:long with multiplehairpins
– Imperfect internal sequencecomplementarity
 CleavedbyDroshainto pre-miRNA
– PrecursormiRNA:~70nt imperfect hairpins
– Exportedfrom nucleus
 CleavedbyDicerinto maturemiRNA(21-25nt)
Novina and Sharp,2004

27. Hannonet.al.,2004

28. RNAi Function
• To regulate expression of protein coding
genes.
• To mediate resistance to both enogenous
and exogenous pathogenic nucleic acid.
• to used experimentally for blocking gene
expression.

30. HIV levels can be reduced by 30-50 fold by siRNA!!!

31. Trait Host Application
Enhanced nutrient content Tomato increased concentration of
caretenoids, hagher
flavonoid and β carotene
Wheet, sweet potato, maize increased levels of amylose
for digestion
canola, peanut increaesed oleic content
reduced production of
lachymatory factor synthase
onion tearless onion
reduced alkaloid production Coffee decaffeinated coffee
Opium poppy Production of
nonnarcotic alkaloid,
instead of morphine
Ethylene senstivity tomato early ripening
Reduced allergenicity peanuts allergen free peanuts

32. References
• RNAi technology: the role in development of Abiotic stress-tolerent crops, Tushar Khare*, Varsha
Shriram*, Vinay Kumar*, Biochemical, Physiological and Molecular Avenues for combacting abiotic
stress in plants, Elsevier 2018.
• MicroRNAs in model and complex organisms, Hikmet Budak, Baohong Zhang, Funct Inegr
Genomics, springer 2017 17:121-124.
• Plant small RNAs: the essential epigenetic regulators of gene expression for salt-stress responses
and tolerance, Vinay Kumar, Tushar Khare, Varsha Shriram, Shabir H. WAni, Plant cell Rep, Springer
2018 37:61-75.
• MicroRNAs as potential targets for abiotic stress tolerence in plants, Varsha Sriram, Vinay Kumar,
Rachayya M. Devarumath, Tushar Khare, Shabir H. Wani, Frontiers in plant sciences, 2016, Vol: 7
Article 817.
• RNA interference: Historical Overview and Significance, Montgomery MK. Methods Mol Biol. 2004.
• www.ncbi.nim.nih.gov/probe/docs/techrnai/
• RNA interference: Biology, Mechanism, and Applications, Neema Agarwal, P.V.N. Dasaradhi and
Sunil K. Mukharjee, MMBR.
• www.umassmed.edu/rti/biology/how-rnai-works-/
• A breif history of RNAi: the silence of the genes, George L. Sen and helen M. Blau, FASEB Journal,
2006.