Rna interfernce ppt

about rna interference

1. RNA interference
Presented by:
C.SWORNA KUMARI
M.PHIL BIOTECHNOLOGY

2. Outlines
Introduction Introduction
RNA silencing
Definition of RNA interference
Discovery of RNAi
Mechanism of RNA interferenceMechanism of RNA interference
Applications of RNA interferenceApplications of RNA interference
Therapeutic applications
Other applications

3. RNA silencing
Several terms are used to described RNA silencing;
usually there are three phenotypically different but
mechanistically similar phenomena:
1.Cosuppression or post-trascriptional gene silencing
(PTGS) in plants
2.Quelling in fungi
3.RNA interference in animal kingdom

4. Definition
RNA interference (RNAi) is a mechanism that inhibits
gene expression at the stage of translation or by
hindering the transcription of specific genes.
RNAi targets include RNA from viruses and
transposons.

5. What is RNA interference (RNAi)?
“The Process by which dsRNA silences gene
expression...” Degradation of mRNA or translation
inhibition

6. Need for interference
Defense Mechanism
Defense against Infection by viruses, etc
As a defense mechanism to protect against transposons and
other insertional elements
Genome Wide Regulation
RNAi plays a role in regulating development and genome
maintenance.
30% of human genome regulated

7. Nobel prize winners in the C. elegans
field
Sidney Brenner
John Sulston
Robert Horvitz
Andrew Fire
Craig Mello

8. RNAi was found to work in many
diverse species
Fungi
Trypanosomes
Insects
Zebrafish
Mice

9. Mechanism of RNAi
RNA i
( RNA Interference )

10. In Interference
RNA
siRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding
genome
RISC:
RNA induced Silencing Complex, that cleaves mRNA
Enzymes
Dicer : produces 20-21 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin in to form pre miRNA; which is
later processed by Dicer

11. siRNAs
Small interfering RNAs that have an integral role in the
phenomenon of RNA interference (RNAi), a form of post-
transcriptional gene silencing
RNAi: 21-25 nt fragments, 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:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs

12. Generation of small interference RNA

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

14. Illustration of miRNA processing

15. 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.
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)

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

17. Dicer’s domains
1 4 32 2
Dicer is a ribonuclease (Rnase III family) with 4 distinct domainsDicer is a ribonuclease (Rnase III family) with 4 distinct domains:
1. Amino-terminal helicase domain
2. Dual Rnase III motifs in the carboxy terminal segment
3. dsRNA binding domain
4. PAZ domain (110-130 amino-acid domain present in protein like
Argo, Piwi..);it is thought to be important for protein-protein
interaction

18. 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 argonaute proteins
which cleave the target
mRNA strand.

19. RNA i
( RNA Interference )
STEPS INVOLVED IN
RNA INTERFERENCE

20. RNA interference
RNA interference (RNAi) is a biological process in
which RNA molecules inhibit gene expression,
typically by causing the destruction of specific mRNA
molecules. Historically, it was known by other names,
including co-suppression, post transcriptional gene
silencing (PTGS), and quelling.

21. STEP 1
•dsRNA is processed
into sense and
antisense RNAs
•21-25 nucleotides in
length
•have 2-3 nt 3’ overhanging
ends
•Done by Dicer (an RNase
III-type enzyme)

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

23. Step 3
the antisense
siRNAs act as
guides for RISC to
associate with
complimentary
single-stranded
mRNAs.

24. Step 4
RISC cuts the
mRNA approximately
in the middle of the
region paired with
the siRNA
The mRNA is
degraded further

25. What are sense and antisense
RNA?
Messenger RNA
(mRNA) is single-
stranded, called
"sense" because it
results in a gene
product (protein).
5´   C U U C A  3´     mRNA
3´   G A A G U  5´     Antisense RNA

26. What are sense and antisense
RNA?
Antisense
molecules
interact with
complementary
strands of
nucleic acids,
modifying
expression of
genes.
5´   C U U C A  3´     mRNA
3´   G A A G U  5´     Antisense RNA

27. Applications of RNAi
RNA i
( RNA Interference )

28. Therapeutic uses of RNAi
Hematology (blood)
Oncology (cancer)
Stem cell biology
Infectious diseases

29. Hematology (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

30. Oncology (cancer)
Targeting of oncogenes
Dominant mutant oncogenes, amplified oncogenes, viral
oncogenes
Define role of signaling molecules in tumor-creation
Improvement efficacy of chemotherapy and
radiotherapy
Tumor regression through creation of potentially new
mode of chemotherapy

31. Stem cell biology
Mouse research
Knock out tumor-suppression gene in mouse embryonic
stem cell
Observe tumor phenotype
Positive correlation between extent of Trp 53
(suppression gene) inhibition and severity of disease

32. 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

33. 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

34. Silencing genes in HIV
AIM:
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.

35. 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

36. 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

37. 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

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

39. Gene Redundancy
In many cases, eliminating the expression of a single gene in
higher eukaryotes can be tolerated even if that gene product
functions in a critical pathway. This is because many critical cell
functions are accomplished by more than one gene product.
When one gene product is eliminated, the redundant gene
product compensates to allow the cell or animal to survive.
Identifying redundant genes could be achieved by co-transfecting
siRNAs and assaying for a given phenotype.
Evaluating each of the candidate genes alone to ensure that they
only cause the cell cycle defect when reduced in combination
with the target gene would help pinpoint the most likely redundant
gene

40. RNA interference characteristics
dsRNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the dsRNA and the target gene/mRNA is
required
Targeted mRNA is lost (degraded) after RNAi
The effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA

41. Advantage of RNAi
Downregulation of gene expression simplifies "knockout"
analysis.
Easier than use of antisense oligonucleotides. siRNA more
effective and sensitive at lower concentration.
Cost effective
High Specifity
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

42. Importance of RNAi
Powerful for analyzing 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
Agriculture

43. Thank you