gene silencing

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Slide 1: 

GENE SILENCING Kirti Khuntwal MTech. Bio-Informatics

From DNA to Protein : 

From DNA to Protein Transcription Process where information held in the DNA is transferred to RNA. Translation Conversion of RNA to amino acid sequence of protein.

Gene Silencing : 

Gene Silencing Gene silencing – “Switching off” of a gene ,by a machinery in the cell. Epigenetic process of gene regulation. Silencing is a position effect . Genes are silenced at either the transcriptional or post-transcriptional level. Transcriptional gene silencing - Result of modifications of either the histones or DNA. e.g.:- Silencing at the yeast telomere. Post-transcriptional gene silencing -Result of the mRNA of a particular gene being destroyed or blocked. A common mechanism of PTGS is RNAi.

Gene silencing by modification of Histones and DNA : 

Gene silencing by modification of Histones and DNA Modification of nucleosomes alter the accessibility of the gene to the transcriptional machinery and regulatory proteins. Heterochromatin is commonly involved in gene silencing, and affects large sections of DNA. E.g.:- the telomere, silent mating -type locus and rDNA gene in the budding yeast S.cerevisae Methylation of particular DNA sequences can also silence transcription in many eukaryotes. E.g. :-the human H19 gene

Silencing at the yeast telomere. : 

Silencing at the yeast telomere.

RNA Interference (RNAi) : 

RNA Interference (RNAi)

History and Discovery : 

History and Discovery 1990 Jorgensen : To deepen the pigmentation in Petunias introduced transgenes homologous to endogenous genes for the enzyme chalcone synthase. Both the endogenous and the transgenes were down regulated. Co-suppression of gene expression.

Slide 9: 

Contd.. 1995 Guo and Kemphues: Found that injection of either antisense or sense RNAs in the germline of C. elegans was equally effective at silencing homologous target genes. 1998 Craig C. Mello and Andrew Z.Fire: Extended the above experiments. A combination of sense and antisense RNA (dsRNA) was 10 times more effective than single strand RNA. Nobel Prize in Physiology or Medicine in 2006 for their discovery that dsRNA triggers the suppression of gene activity in a homology- dependent manner. They coined the term RNA interference for the unknown mechanism.

RNAi was discovered in C.elegans : 

inject worms with dsRNA corresponding to a gene involved in wiggling (unc-22) RNAi was discovered in C.elegans

RNAi was discovered in C.elegans : 

conclusion: dsRNA triggers potent and specific gene silencing inject worms with dsRNA corresponding to a gene involved in wiggling (unc-22) RNAi was discovered in C.elegans

Gene-Silencing in Some Species : 

Gene-Silencing in Some Species

What is RNA Interference : 

What is RNA Interference Post-transcriptional gene silencing (PTGS). Process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of mRNA. RNAi is highly gene-specific. There are 2 pathways - Exogenous (A viral RNA genome or laboratory manipulated) mediated by siRNA. Endogenous (originating in the cell) mediated by miRNA.

Mechanism of RNAi : 

Mechanism of RNAi 2 step mechanistic model: Initiation step Generation of siRNAs Effector step Degradation of target mRNA

Initiation Step : 

Initiation Step ATP ATP ADP + ppi ADP + ppi DICER KINASE RdRP

Effector Step : 

Effector Step siRNA binding siRNA unwinding RISC activation

Major Components in RNAi : 

Major Components in RNAi Dicer RNA-Induced Silencing Complex (RISC) Small interfering RNA (siRNA) miRNA

Slide 18: 

Dicer Member of RNAseIII family of enzymes. Recognize and process dsRNA into siRNA. Dicer family proteins are ATP-dependent nucleases. Dicer homologs exist in many organisms including C.elegans, Drosophila, yeast and humans.

Dicer : 

Dicer Amino-terminal helicase domain. 2 RNaseIII catalytic domain. dsRNA binding domain. PAZ domain.

Dicer acts as dimer : 

Dicer acts as dimer

RNA-Induced Silencing Complex (RISC) : 

RNA-Induced Silencing Complex (RISC) Large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA. The active component of RISC are endonucleases called argonaute proteins . The strand binding to the argonaute protein - ‘guide strand’. The other ‘anti-guide strand or passenger strand is degraded during RISC activation. The strand chosen is the one whose 5’ end is least paired to its complement. The process is ATP independent.

RISC : 

RISC 2 RNA binding proteins RNA/DNA Helicase Translation Initiation Factor RNA-Dependent RNA Polymerase (RdRP) Transmembrane Protein

Small Interfering RNA (siRNA) : 

Small Interfering RNA (siRNA) 21-25 nucleotide dsRNA with 2-nt 3’ overhangs on either end. Produced in vivo by cleavage of dsRNA or exogenously introduced in the cell. Amplification by an RNA-dependent RNA polymerase (RdRP) may occur. Incorporated into the RISC guiding it to mRNA A single base pair difference between the siRNA template and the target mRNA is enough to block the process.

siRNA : 

siRNA 5’ 3’ 5’ 3’

miRNAs /stRNAs : 

Micro/small temporal RNAs – ssRNA molecules of 21-23nt in length. Genomically encoded non-coding RNAs . Approx. 500 miRNAs are found in mammalian cells and 30% of all genes are regulated by miRNAs. Some are expressed in all cells and at all developmental stages and others have a more restricted spatial and temporal expression pattern. 30% of the miRNAs found in worms have close homology in flies and/ or mammalian genomes. miRNAs /stRNAs

Processing of miRNA : 

Processing of miRNA

Significance of RNAi : 

Significance of RNAi Protects against viral infection. Secures the genome stability by keeping mobile elements silent. Repress protein synthesis and regulate development of organism. Keep chromatin condensed and suppress transcription. Experimental tool to elucidate the function of any gene. Biotechnology – engineering of food plants. Useful approach in future gene therapy.

siRNA design : 

siRNA design Target Sequence- 21-nucleotides long , 50-100 bp downstream from start codon (AUG) Search for seq. motif AA(N19). Avoid sequences with > 50% G+C content. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats. Avoid sequences that share a certain degree of homology with other related or unrelated genes.

Slide 32: 

from Mittal, Nature Rev.Genet. 5, 355 (2004) The Design of Optimal siRNAs 21 nt RNA that contains 2 nt 3’- overhangs and phosphorylated 5’-ends Lower stability at the 5’-end of the antisense terminus Low stability in the RISC cleavage site Low secondary structure in the targeted region of the mRNA

Vectors expressing siRNAs : 

Vectors expressing siRNAs U6 H1 siRNA

Vectors expressing siRNAs : 

Vectors expressing siRNAs U6 Sense sequence Anti-sense sequence Hair-pin loop

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RNAi can be induced by

How long does RNAi last? : 

How long does RNAi last? Silencing caused by microinjection (synthetic siRNA) lasts for several days and appear to be transferred to daughter cells, but eventually diminish. Cells transfected with siRNA expression vector experience steady, long-term mRNA inhibition.

RNAi Tools and Products : 

RNAi Tools and Products siRNA Delivery GeneSuppressor RNA Interference Kit Targefect-siRNA Transfection Kit TransIT -TKO Transfection Reagent siRNA localization Label IT siRNA Tracker Kits siRNA expression PCR-Based siRNA Expression Systems

siRNA Therapeutics : 

siRNA Therapeutics “Instead of trying to stop a flood by mopping the floor, with RNAi we can stop the flood by turning off the faucet.”

RNAi as a powerful therapeutic drug : 

RNAi as a powerful therapeutic drug Exquisitely selective like the fabled “magic bullet”. May work synergistically with other drug and treatment regimes. Endogenous pathway so allows the development of safe and efficacious drugs. Potent- very low concentrations of siRNA are required. The clinical applications appear endless.

Delivery,Delivery,Delivery…. : 

Delivery,Delivery,Delivery…. Charged oligonucleotide cannot penetrate cellular membranes. dsRNA-dependent protein kinase response Unmodified RNA rapidly excreted or degraded by RNase.

Slide 42: 

from Dykxhoorn and Lieberman, Cell 126, 231 (2006) Delivery of siRNA for Therapy siRNA is not taken up by most mammalian cells Cholesterol-conjugated siRNA is taken up by the LDL receptor siRNA bound to targeted antibody linked to protamine can achieve cell-specific siRNA delivery

Therapeutic siRNAs : 

Therapeutic siRNAs (Sioud, 2004)

Therapeutic siRNAs : 

Therapeutic siRNAs

Slide 45: 

THANKS

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