GENE SILENCING

1. GENE SILENCING By Dr.S.K.Sarangi Dept.of MB & BT Jnanabharathi Campus Bangalore University Bangalore-560 056

2. Gene Silencing: • Definition: • It is the epigenetic process of gene regulation that describes the “switching off” of a gene by a mechanism other than the genetic modification. • It also protects organism’s genome from transposons and viruses. • Types: • 1] Transcriptional Gene Silencing • 2] Post-transcriptional Gene Silencing

3. TRANSCRIPTIONAL GENE SILENCING • Brought out at the gene level. • It makes the gene inaccessible to transcriptional machinery (RNA • Polymerase, Transcriptional factors etc.). • By modification of Histones and associated induction of • heterochromatin formation. • By region specific hypermethylation of DNA (mainly the CpG islands). • Effected through RITS (RNA Induced Transcriptional Silencing). • RITS is a complex which contains small RNAs, Argonaute (a nuclease) • and two proteins (Chp 1,a chromatin associated protein & Tas 3,a novel • protein). • RITS disrupts histone methylation and centromere formation to down • regulate the gene function.

4. Contd. • RITS complex can also associate with specific si RNAs that are • complementary to the local genes. • Some siRNAs bind to histones and degrade any nascent • mRNA formed, thereby inhibiting transcription. • miRNAs are found to affect gene expression via Histone • Modification and DNA Methylation of the target gene’s promoter. • miRNAs also target transcription factors and co-activators and • result in TGS.

5. Effected through the mRNA of a particular gene being • destroyed or blocked. This is known as RNA interference • (RNAi) pathway. • Two mechanisms exist in RNAi pathway such as: • a)Cleavage mechanism(siRNA & miRNA) • b)Bypass mechanism (Antisense RNA) Post Transcriptional Gene Silencing

6. DISCOVERY OF RNAi Nepoli et.al (1990) first to observe in flowers of Petunia. But couldn’t explain the mechanism. 1998, Andrew Fire of Carneign Institute of Washington and Creig Mello of University of Massachusettes were the first to explain the RNAi mechanism in Caenorhabditis elegans. They received Nobel Prize for their discovery in 2006. Creig Mello and Andrew Fire

7. Cleavage Mechanism:It is brought about by 2 types of RNAs 1) Small interfering RNA (siRNA) »Long double stranded RNA (dsRNA > 200nt.) coming from exogenous sources, mainly viruses or transposons, enter a cellular pathway referred to as RNA interference (RNAi). »Long ds RNA gets digested to small interfering RNAs (siRNA, 20- 25 nt.) by an RNase III like enzyme called Dicer in the cytoplasm. » These siRNAs assemble into RISC (RNA Induced Silencing Complex),having Argonaute (a nuclease).

8. contd. » The RISC unwinds the double stranded siRNAs to single strands by digesting the cognate strand. » The single stranded siRNA (Guide strand) then is guided by the RISC to the complementary mRNA and binds to it. » The target mRNA is then cleaved by Argonaute and destroyed. » Hence the gene product (the protein) is not synthesized, resulting in Gene Silencing.

10. DISCOVERY OF miRNA Victor Ambros, Gary Ruvkun, Rosalind C. Lee in Harvard University reported the first small regulatory RNA (ribonucleic acid) microRNAs that they identified in their genetic studies of the development of C. elegans

11. 2) Micro RNA (miRNA) אmiRNAs are mostly indigenous. אEncoded by long RNA coding genes. א They are about 1000nt. long and called Primary miRNA (Pri-miRNA). א Form hairpin loops- existing as dsRNA. א They form a Microprocessor complex in the nucleus, consisting of Pri-miRNA, Drosha (RNase III type enzyme) and Pasha (ds RNA binding protein). א Drosha, cleaves the Pri-miRNA to small double stranded RNA molecules of about 70nt.long-called the pre-miRNA. א Introns, sometimes directly give rise to Pre-miRNA in the nucleus- bypassing microprocessor complex formation.

12. Pre-miRNA then exported to the cytoplasm (Exportin 5) and cleaved into small • pieces of dsRNAs of 21-23nt long by Dicer (RNase III type enzyme), called • miRNA. • In the cytoplasm, the miRNA assemble to RISC (RNA Induced Silencing Complex) • that contains Argonaute (a nuclease). • The RISC unwinds the double stranded RNA and the cognate strand is digested by • the Argonaute. • The miRNA is then processed to single stranded mature miRNA of about 21- • 23nt. long. • The mature miRNA is then guided by the RISC towards the untranslated mRNA and • binds to it. • In plants the miRNA is completely complementary to the mRNA- Hence binds and • cleaves the mRNA, similar to siRNA. • But in Animals, miRNA is partially complementary- Hence binds to a portion of the • mRNA and blocks the translation. Contd.

13. Functions of miRNA: • Gene Silencing through TGS & PTGS. • Regulate Immune Systems. • » Development of Lymphocytes. • » Generation of Cytokines and Antibodies. • »Proliferation of Neutrophils. • »Impairment of ‘T’-Cell development. • Pathogenesis of Cancers through Cell proliferation and • apoptosis. • Uncontrolled autoimmunity.

15. SIMILARITIES AND DIFFERENCES BETWEEN siRNA AND miRNA DIFFERENCES siRNA derived from double strand product of a virus or transposon or double strand RNA provided by a researcher, targets same transcript from which it arose. Where as miRNA encoded by a conventional segment of genome by individual or cluster of genes, their own genes or derived from introns of genes. siRNA bring about gene silencing by degradation of mRNA, miRNA bring about gene silencing by blocking translation by binding to 3’ untranslated region. • SIMILARITIES • SIZE: miRNA 21-23 nucleotides in length, siRNA 20-25nucleotides in length. Hence, have same size range. • Produced by similar processing machinery. Therefore, considered as “cousins” • Both involved in gene silencing.

16. b) Bypass mechanism (Antisense RNA) א It is brought about by antisense RNA. א Antisense RNA is a single stranded RNA that is complementary to the sense mRNA. א Sense mRNA is capable of translation which is encoded by the antisense DNA strand and has the similar sequence as the sense DNA strand. א By producing a transgenic organism which could transcribe the sense DNA strand, an antisense RNA can be produced. א Thus the antisense strand having complementary base sequences , base pairs with the sense mRNA strand and forms a double stranded RNA. א The double stranded RNA is not translatable because it is not recognized by the ribosome. א Hence the translation of that particular protein is blocked. א Antisense RNA may be produced naturally (in some cases) or may be synthesized artificially and introduced to the cell. Eg:- Flavr Savr Tomato, Papaya.

18. APPLICATIONS OF RNAi AGRICULTURE Transgenic papaya- resistant to ring spot viruses Arabidopsis thaliana resistant to mosaic virus. Cotton- its own insecticide “gossypol” rendered effective against cotton bullworm. Onion – tearless onions produced by shutting down lachramatory factor synthase gene and re-directing sulphur towards making nutritive and flavoring compounds. Coffee- decaffinated coffee plants produced by knocking down threobrominesynthase gene. Transgenic tobacco- with less carcinogen (nicotin) levels produced. World’s first blue rose produced by Florigem and Suntory companies.

19. APPLICATIONS OF RNAi MEDICINE • Leukemia- BCR-ABL fusion genes converted back to normal form. • Hepatitis and influenza- using complememtary RNA in lab monkeys. • Herpes- vaginal application of siRNA in mice. • HIV • Respiratory Syncytial virus (RSV)- inhalable antiviral RNAi preparation. • SARS corona virus- intranasal administration of siRNA in monkeys. • Age related macular degeneration- siRNA injected directly into eyeball. • Huntington’s disease- mutant huntingtin protein mediated by adeno associated viral vector carrying siRNA. • Bad cholesterol- siRNA against APOB mRNA.

20. DRAW BACK OF RNAi IN TREATMENT The stem-loopsecondary structure of a pre-microRNA from Brassica oleracea. • RNAi may prove to be ineffective against viruses because these pathogens tend to mutate rapidly. Mutations result in change in genome sequence, leading to production of mRNAs that are no longer fully complementary to the therapeutic siRNA

21. Thank You