The document provides an overview of RNA interference (RNAi), detailing its mechanisms, discovery, and applications in gene silencing within eukaryotic organisms. It discusses the distinctions between small interfering RNAs (siRNAs) and microRNAs (miRNAs), their roles in regulating gene expression, and their implications in cancer pathogenesis and agricultural protection. RNAi is emphasized as a vital tool for controlling gene expression and addressing various biological challenges.

1. DONE BY: HADIAH BASSAM AL MAHDI
M A S T E R O F S C I E N C E
G E N E T I C S D E P A R T M E N T , F A C U L T Y O F S C I E N C E
RNA INTERFERENCE

2. OUTLINE
 Introduction
 RNA silencing
 Definition of RNA interference
 Discovery of RNAi
 Mechanism of RNA interference
 siRNA
 miRNA
 Expolotion of RNA interference
 Conclusion

3. INTRODUCTION

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

5. Definition
RNA interference (RNAi) is a mechanism
that inhibits gene expression at the stage
of translation or by hindering the
transcription of specific genes.

6. Why do we 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. Discovery Of RNAi
 Making petonia flower with deeper purple color by
inserting extra copies of the gene into cell resulted in
paler flowers
 Inserting antisense RNA of par-1 gene to block it action
 The sense RNA which was used as control did the same job
 RNA from other genes did not affect par-1 gene
 2006 Nobel prize for the discovery that it is the dsRNA
(caused by contamination) which
blocked the transcription

8. MECHANISIM OF RNAi

9. RNAi Mechanism
 RNA interference (RNAi) acts in 3 ways:
1. Inhibit transcription by silencing the gene
2. Inhibit translation of mRNA
3. Destruct mRNA

10. where do these RNAi come from?
 Small interfering
RNA (siRNA) are
made from dsRNA
precursors
 microRNA (miRNA)
are encoded by genes

11. 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
 The catalytic component of the RISC complex
include Argonaute
 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

12. The mechanism of siRNA :
 Long dsRNA .
Dicer
 siRNA into two single-
stranded RNAs (ssRNAs)
 Guide (siRNA) enters the cell.
RISC + Argonaute
 Single stranded siRNA binds
to its target mRNA.
 The mRNA is now cut and
recognized as abnormal by the
cell.

13. Synthesis of miRNA
Gene encoding miRNA (nucleus)
(transcription)
Pri-miRNA
(Drosha)
Pre-miRNA (get out to cytoplasm)
(Dicer)
Mature-miRNA
 (RISC)
miRNA binds to its target mRNA
and Degraded

14. • Drosha cuts 11 bp away from dsRNA-ssRNA
junction
• Dicer is needed to generatemature miRNA from
pre-miRNA

15. miRNA siRNA
 Precursor: 70 nt shRNA
 Imperfect pairing required
 Function: mRNA cleavage
 Nearly conserved
 Encoded by own genes
(endogenous source )
 Precursor: Long dsRNA
 perfect pairing required
 Function: mRNA cleavage
 Not conserved
 Encoded by transposon or
viruses (exogenous source)
miRNA vs siRNA

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17. Expolotion OF RNAi

18. Expolotion of RNAi
 Protection against transposons & viruses
 Faster identification of gene function
 Role in regulating gene translation
 Role in cancer pathogenesis
 Agriculture

19. Protection against transposons & viruses
 45% of human genome is DNA originated from
transposons
 RNAi machinery silence transposons by recruiting
histone modifiers
 Mutations affecting RNAi machinery could reactivate
transposons
 RNAi machinery 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. Role in regulating gene translation
 Huntington’s disease
 Disease caused by toxin protein,
that affects more than 30,000
people.
 Gene silencing aims to reduce
the production of the mutant
protein by shut down the
expression of the gene coding
for the aberrant protein

21. Role in cancer pathogenesis
 miRNA are classified as:
1. Oncogenic: their increase expression lead to cancer
1. Tumor suppressor: their absence increase cancer
 Half of the identified miRNA (~300) are located at
chromosomal regions disrupted by rearrangement
in cancer pathogenesis

22. Oncogenic miRNA
(miR-17-92)
Tumor suppressor
miRNA (miR-15 & miR-16)
 Located in 13q31
 Expression of miR-17-92 increased in
small cell lung cancer
 It target RB2 and E2F1 responsible
for regulating cell cycle (tumor
suppressor genes)
 Located in 13q14
 Deletion of miR-15 & miR-16 will reduce
their expression  chronic lymphocytic
leukemia (CLL)
 miR15+16 BCL2apoptosis
 Deletion of miR15+16
BCL2apoptosis
Role in cancer pathogenesis

23. CONCLUSION

24. Conclusion
 Eukaryotes use dsRNA as an agent of gene silencing, in a process called RNAi
 Special enzymes (Drosha and, most generally Dicer) recognize dsRNA and generate
from that short (21-22 nucleotide) RNAs that are the active species for gene
silencing.
 The dsRNAs that give rise to siRNAs can arise from various sources ranging from
infecting viruses.
 siRNAs and miRNAs act in essentially the same way. They are incorporated into a
machine called RISC.
 miRNAs have also been associated with cancer, with some miRNAs being classified
as tumor suppressors and others as oncogenes.
 RNAi has become a regulator tool and is particularly useful in systems.

25. Thank You