RNA interfernce

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference.

1. Done by: Hadiah Bassam Al MAHDI Master OF SCIENCE GENETICS DEPARTMENT, FACULTY OF SCIENCE 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: post-trascriptional gene silencing (PTGS) in plants Quelling in fungi 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: Inhibit transcription by silencing the gene Inhibit translation of mRNA 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 ) G uide ( siRNA ) enters the cell . RISC + Argonaute S ingle 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

16. https:// www.youtube.com/watch?v=2dL7Sh_udKs

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: Oncogenic: their increase expression lead to cancer 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.

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