Gene silencing

Gene silencing

1. Gene silencing Prepared by: Zeinab Klaab Submitted to: Prof. Saad ALqhtani ZOO651

2. Contents: Introduction Short history of gene silencing Gene silencing methods Applications of RNAi

3. Introduction Gene silencing is a technique that aims to reduc e or eliminate the production of a protein from it’s corresponding gene. It generally describe the “switching off” of a gene. It occurs when RNA is unable to make a protein during translation. Gene silencing is same as gene knockdown but is totally different from gene knockout.

4. Short history of gene silencing 1990 Jorgensen : • To deepen the pigmentation in endogenous genes resulted in plants with both gene suppressed called co suppression . 1995 Guo and Kemphues : • Injection of either antisense or sense RNAs in the germline of C. elegans was equally effective at silencing at homologous target genes. 1998 Mello and Fire: • Extension of above experiments, combination of sense and antisense RNA(=dsRNA) was 10 times more effective than single strand RNA. • The discovery of the mechanism of RNA interference by ds RNA by prof. Fire and prof. Mello in 1998, gave them the Nobel prize in 2006 .

5. Types of Gene silencing Genes are regulated at either the transcriptional level or post-transcriptional level , therefore silencing can be induced either at transcriptional level or posttranscriptional level . There are mainly two types of gene silencing 1 . Transcriptional gene silencing 2 . Post transcriptional gene silencing Post transcriptional gene silencing Transcriptional gene silencing e.g. Antisense RNA technology1. RNAi technology e.g. Genomic Imprinting Position effect

6. Transcriptional gene silencing • Genomic imprinting: Genomic imprinting or parental imprinting occurs when a gene inherited from either the father or the mother has been permanently inactive in that parent and passed on to offspring in the same inactivated condition . The inactivated genes has not mutated – the DNA sequence is normal . It is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.).

7. Inactivation caused by methylation of DNA in the regulatory sequences of the inactivated gene This results in the silencing of an allele in either male ( paternally imprinted ) or female ( maternally imprinted ) gametes .

8. For example- Insuline like growth factor gene(Igf2) in mice • The product of these gene is required for normal growth in the embryo • If paternal allele is highly methylated and silence, only maternal allele is expressed, results shows that dwarfing mutant mice produced

9. • Position effect : Position effect is the effect on the expression of a gene when its location in a chromosome is changed , often by translocation . This has been well described in Drosophila with respect to eye colour and is known as position effect variegation (PEV ). For example eye colour (w+ shows red pigment) in Drosophila

10. Post transcriptional gene silencing Anti sense RNA technology : • Antisense RNA has the opposite sense to m-RNA. • The presence of complimentary sense and antisense RNA in the same cell can lead to the formation of a stable duplex which interferes with gene expression at the level of RNA processing (translation). • This technology widely used in plants The ability of exogenous or sometimes endogenous RNA to suppress the expression of the gene which corresponds to the m-RNA sequence

11. RNAi (Interference)Technology RNA interference (RNAi ): • Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA . • A selective gene knockdown phenomenon.

12. Overview of RNAi : • During RNAi , Double-stranded RNAs cut into short double-stranded RNAs, i ( interfering) RNAs, by an enzyme called Dicer . These then base pair to an mRNA through a dsRNA-enzyme complex ( RISC). This will either lead to degradation of the mRNA strand . • Highly specific process • So far only been seen in eukaryotes • Evidence 30% of genome is regulated by RNAi • RNAi pathway guided by, There are three types of dsRNA produed and they leads to RNAi pathway: small interfering RNAs (siRNAs) generated via processing of longer dsRNA microRNAs (miRNAs) that are generated via processing of stem loop precursors short hairpin RNAs (shRNA) that are generated via hair pin structure

13. Mechanism of RNAi 1- RNA A- siRNA: dsRNA 21-22 bp. B- miRNA: ssRNA 19-25 bp. Encoded by non protein coding genome 2- RISC: RNA induced Silencing Complex, that cleaves mRNA Enzymes 3- Dicer : produces 20-21 bp cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre miRNA in nucleus ; which is later processed by Dicer in cytoplasm .

14. 1- (A): siRNAs Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of posttranscriptional gene silencing RNAi: 21-25 bp fragments, which bind to the complementary portion of the target mRNA and tag it for degradation  Can be exogenously (artificially) introduced into cells by transfection 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

15. Generation of small interference RNA

16. 1- (B): miRNA Originate from capped & polyadenylated full length precursors ( pri -miRNA) Hairpin precursor ~70 bp (pre-miRNA) Mature miRNA ~22 bp (miRNA) Illustration of miRNA processing

17. 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 .

18. RNAse III-like dsRNA-specific ribonuclease It is able to digest dsRNA into uniformly sized small RNAs ( si RNA ) Dicer family proteins are ATP- dependent nucleases . Loss of dicer→loss of silencing processing in vitro DICER’s domain : Dicer is a ribonuclease ( RNAse III family) with 4 distinct domains : 1) Amino-terminal helicase domain 2) Dual RNAse III motifs in the carboxyl terminal segment 3) dsRNAbinding domain 4) PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi ..); it is thought to be important for potein -protein interaction 2- Dicer

19. 3- RNA-Induced Silencing Complex (RISC) RISC Nuclease complex composed of both protein and RNA RISC is a large (~500-kDa) RNA-multi protein complex, which triggers mRNA degradation in response to s iRNA Unwinding of double- stranded siRNA by ATP independent helicase. The active components of an RISC are endonucleases called argonaute (Ago ) proteins which cleave the target mRNA strand

20. STEPS INVOLVED IN RNA INTERFERENCE 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) Step 2 The siRNAs associate with RISC and unwind

21. STEPS INVOLVED IN RNA INTERFERENCE Step 3 the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs. Step 4 RISC cuts the mRNA approximately in the middle of the region paired with the siRNA The mRNA is degraded further

22. Advantages of RNAi Downregulation of gene expression "knockdown" analysis . Easier than use of antisense oligonucleotides . siRNA more effective and sensitive at lower concentration. Cost effective Blocking expression of unwanted genes Useful approach in future gene therapy Disadvantages of RNAi High pressure injection can cause significant damage to the normal tissues and organs Liposomes/cationic encapsulated siRNA may also be toxic to the host and may cause immune responses .

23. Therapeutic applications of RNAi : Cancer : The goals for RNAi approaches for cancer therapy are therefore to knock out the expression of a cell cycle gene and/or an anti-apoptotic gene in the cancer cells thereby stopping tumor growth and killing the cancer cells Infectious Diseases : suppression of HIV-1 replication in human cells transfected with siRNA. Neurodegenerative Disorders: Alzheimer's disease, Parkinson's disease and Huntington's disease are examples of relatively common age-related neurodegenerative disorders

24. The U.S. Food and Drug Administration has approved infusions of Patisiran ( Onpattro , Alnylam Pharmaceuticals) to treat peripheral nerve disease (polyneuropathy) caused by hereditary transthyretin-mediated amyloidosis ( hATTR ). It is the first-ever approval of a new class of drugs, small interfering ribonucleic acid (siRNA), used to treat rare and genetic diseases.

25. Polyneuropathy is a rare, debilitating, and often fatal genetic disease affecting approximately 50,000 people worldwide and it is characterized by the buildup of abnormal amyloid protein in peripheral nerves, the heart and other organs. “This approval is part of a broader wave of advances that allow us to treat disease by actually targeting the root cause , enabling us to arrest or reverse a condition, rather than only being able to slow its progression or treat its symptoms,” FDA Approves Patisiran , First-Ever RNA Interference Therapeutic Approved for Clinical Use (2018)

26. Abstract: Fitusiran is an RNA interference therapeutic that targets a ntithrombin (AT) in the liver and interferes with AT translation by binding and degrading messenger RNA-AT , thereby silencing AT gene expression and preventing AT synthesis . Preclinical in vitro and in silico studies indicate improvement in thrombin generation in rare bleeding disorder plasmas, suggesting potential therapeutic benefit.

27. Hemophilia is an X-linked bleeding disorder resulting from a deficiency of factor VIII, for hemophilia A, or a deficiency of factor IX, for hemophilia B RNAi utilizes a normal cellular process which results in highly specific gene silencing . The availability of novel therapeutics, in particular siRNA targeting antithrombin (AT) for those with hemophilia, has the potential to reduce bleeding and the burden of treatment, and improve quality-of-life . An investigational RNAi therapeutic targeting antithrombin for the treatment of hemophilia A and B Machin N. and  Ragni MV. (2018 )

28. Site of action of fitusiran on coagulation cascade . Note: Fitusiran is an RNAi therapeutic that targets AT in the liver and interferes with AT translation by binding and degrading mRNA-AT, silencing AT gene expression, and preventing AT synthesis .