RNAi silencing- miRNA and siRNA and its applications.pdf

RNAi silencing- miRNA and siRNA and its applications

1. RNA INTERFERENCE (RNA -I)
DR. MANIKANDAN KATHIRVEL M.SC., PH.D., (NET)
ASSISTANT PROFESSOR,
DEPARTMENT OF LIFE SCIENCES,
KRISTUJAYANTICOLLEGE (AUTONOMOUS),
REACCREDITED WITH "A++" GRADE BY NAAC
K. NARAYANAPURA , KOTHANUR (PO)
BENGALURU 560077
ORCID ID: 0000000270066334

2. INTRODUCTION
RNA i(RNA interference): it is a post transcriptional process triggered by the
introduction of double stranded RNA (ds RNA) which leads to the gene silencing in a
sequence specific manner.
First evidence came from studies on nematode Caenorhabditis elegans. further analysis in
fruit fly drosophila melanogaster.
It is also known as post transcriptional gene silencing / co suppression and quelling. Post
transcriptional gene silencing.
RNAi targets include RNA from viruses and transposons.

4. OVERVIEW
During RNA iDouble-stranded RNAs cut into short double-stranded RNAs, s(small)
i(interfering) RNAs, by an enzyme called Dicer.
These then base pair to an mRNA through a dsRNA-enzyme complex.
This will either lead to degradation of the mRNA strand
•Highly specific process
•Very potent activity
•So far only been seen in eukaryotes
•Evidence 30% of genome is regulated by RNA I

6. MECHANISM

7. IN INTERFERENCE
RNA
•SiRNA-dsRNA21-22nucleotides.
•miRNA-ssRNA19-25nucleotides.
RISC:RNAinducedSilencingComplex,thatcleavesmRNA.
Enzymes
•Dicer-produces20-21ntcleavagesthatinitiateRNAi.
•Drosha-cleavesbasehairpinintoformpremiRNA;whichislaterprocessedby
Dicer.

8. Si RNA
•Small interfering RNAs that have an integral role in the phenomenon of RNA interference
(RNAi), a form of post-transcriptional gene silencing.
•21-25 ntfragments, which bind to the complementary portion of the target mRNA and tag it for
degradation.
•A single base pair difference between the siRNA template and the target mRNA is enough to
block the process.
•Each strand of siRNA has
5’-phosphate termini
3’-hydroxyl termini
2/3-nucleotide 3’ overhangs

9. Mi RNA
•miRNA Originate from capped & polyadenylated full length precursors (pri-miRNA)
•Hairpin precursor ~70 nt(pre-miRNA) Mature miRNA ~22 nt(miRNA)

10. 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 ssRNAthat forms a hairpin secondary structure.
•miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target.
•And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation
serves to downregulate genes pre-transcriptionally (RNA induced transcriptional silencing or RITSRITS)

11. Dicer
•RNase III-like dsRNA-specific ribonuclease
oEnzyme involved in the initiation of RNAi.
oIt is able to digest dsRNA into uniformly sized small RNAs (siRNA)
•Dicer family proteins are ATP-dependent nucleases.
•RnaseIII enzyme acts as a dimer
•Loss of dicer→lossof silencing processing in vitro.
•Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer is a
conserved protein)

12. RISC
•RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA
degradation in response to siRNA.
•Unwinding of double-stranded siRNA by ATP independent helicase.
•The active components of an RISC are endonucleases called argonauteproteins which
cleave the target mRNA strand.

13. STEPS INVOLVED IN RNA INTERFERENCE
STEP 1
•dsRNA is processed into sense and antisense RNAs
o21-25 nucleotides in length
ohave 2-3 nt3’ overhanging ends
oDone by Dicer (an RNase III-type enzyme)

14. STEP 2
The siRNAs associate with RISC
(RNA-induced silencing complex)
and unwind

15. STEP 3
The antisense siRNAs act as guides
for RISC to associate with
complimentary single-stranded
mRNAs.

16. STEP 4
RISC cuts the mRNA approximately
in the middle of the region paired
with the siRNA The mRNA is
degraded further.r

17. APPLICATIONS
Therapeutic uses of RNAi Haematology (blood) Oncology (cancer) Stem cell biology
Infectious diseases
Haematology (blood) Hematologic disorders result from Loss of gene function Mutant
gene function Absent gene function RNAi May be used to create models of disease
processes Could help to develop pharmacologic and genetic therapeutic targets.
Oncology (cancer) Targeting of oncogenes Dominant mutant oncogenes, amplified
oncogenes, viral oncogenes Define role of signalling molecules in tumour-creation
Improvement efficacy of chemotherapy and radiotherapy Tumour regression through
creation of potentially new mode of chemotherapy.

18. Stem cell biology
Mouse research
Knock out tumour-suppression gene in mouse embryonic stem cell
Observe tumour phenotype
Positive correlation between extent of Trp53 (suppression gene) inhibition and severity of disease.
Infectious Diseases
Virus targeting
RNAi inhibit cellular and viral factors of disease
RNA transcriptase is RNAi target
Inhibition of replication
Main goal
Render cells resistant to infectious organisms

19. Hepatitis C
Infects ~200 million people worldwide
Often fatal
2002, Anton McCaffrey and Mark Kay at Stanford University
Injected "naked" RNA strands into the tail veins of mice RNAi treatment controlled the virus
in mice
Silencing genes in HIV
Silence the main structural protein in the virus, p24, and the human protein CD4.
Hit the virus where it counts by eliminating a protein it needs to reproduce or cause
infections.

20. Respiratory infections
RSV ( RESPIRATORY SYNCTIAL VIRUS), infects almost every child by the age of two
Short strands of "naked" RNA
Controlled the virus in mice
Clinical trials are ongoing
Macular degeneration
Macular degeneration is the leading cause of adult blindness
Excess VEGF which leads to sprouting of excess blood vessels behind the retina & obscuring vision.
The new RNAi drugs shut down genes that produce VEGF. The drug can be injected directly into the eye
First clinical trial: 24 patients, launched in 2004.
Two months after being injected with the drug, 6 of the patients had significantly clearer vision
Other patients' vision had at least stabilized
More extensive trials are ongoing

21. Huntington’s disease
Ideal candidate for RNAi therapy
Disease caused by protein, that affects more than 30,000 people in the U.S. alone.
We would want to shut down the expression of the gene coding for the abberant
protein
2004, Beverly Davidson and colleagues at the University of Iowa
Davidson treated mice with Huntington's

22. Other uses of RNAi
Testing Hypotheses of Gene Function
Target Validation
Pathway Analysis
Studying cell division
Gene Redundancy
Functional Screening

23. IMPORTANCE OF RNAi
Powerful for analysing unknown genes in sequenced genomes.
Efforts are being undertaken to target every human gene via siRNAs
Faster identification of gene function
Gene therapy: down-regulation of certain genes/ mutated alleles
Cancer treatments
•knock-out of genes required for cell proliferation
•knock-out of genes encoding key structural proteins.

24. REFERENCES
•https://www.slideshare.net/swormage/rna-interfernce-ppt
•https://www.abcam.com/pathways/rna-interference---a-comprehensive-overview