POST TRANSCRIPTIONAL GENE SILENCING-AN INTRODUCTION.pptx

This video speaks about RNA interference

1. POST TRANSCRIPTIONAL GENE SILENCING (PTGS) Dr ARPITA MISHRA

2. Gene silencing Interruption or suppression of the expression of a gene at transcriptional or translational levels . Two classes: Transcriptional Gene silencing Post Transcriptional gene silencing Post-transcriptional gene silencing (PTGS) is a mechanism that degrades specific messenger RNAs and thereby reduces the expression of a specific gene. PTGS has many names: Co-suppression in plants, Quelling in fungi and RNA interference (RNAi) in animals, In all cases, mRNA is degraded to decrease gene expression.  The process is homology dependant PTGS mechanisms naturally occur in organisms

3. PTGS can be artificially induced by the introduction of double-stranded RNA (dsRNA) A potent tool in functional genomics Is used as a tool to knock down expression of specific genes in a variety of organisms  Therapeutic applications in cancer, infectious diseases and neurodegenerative diseases History Co-suppression was observed for the first time in petunias. In a Genetic Engineering experiment, Rich Jorgensen and colleagues in1990 introduced pigment-producing genes under the control of a powerful promoter into petunia plants The genes introduced were chalcone synthase ChsA transgene or a dihydroflavonol-4-reductase transgene.Both genes encoded proteins involved in the production of anthocyanin pigments. The intention was to deepen the purple colour of petunia flowers. However many flowers appeared variegated or even white Jorgensen named the observed phenomenon " cosuppression ", since the expression of both the introduced gene and the homologous endogenous gene was suppressed

4. RNA –I Fire and Mello first injected dsRNA — a mixture of both sense and antisense strands — into C. elegans . The investigators injected dsRNA corresponding to a 742-nucleotide segment of unc22 gene into either the gonad or body cavity region of an adult nematode unc22 encodes an abundant but nonessential myofilament protein, and the decrease in unc22 activity is supposed to produce an increasingly severe twitching phenotype The injected animal showed weak twitching, whereas the progeny individuals were strong twitchers . Thus it was shown that exogenous introduction of dsRNA corresponding to a gene could result in silencing of the endogenous gene The injection resulted in much more efficient silencing than injection of either the sense or the antisense strands alone. Indeed , injection of just a few molecules of dsRNA per cell was sufficient to completely silence the homologous gene's expression. Furthermore, injection of dsRNA into the gut of the worm caused gene silencing not only throughout the worm, but also in its first generation offspring This process, of PTGS by the introduction of dS RNA was named RNA interference

5. Features of RNA- i Features of the RNA- i mechanism are: The process is initiated by the dsRNA, The target RNA is degraded in a homology dependent fashion, and The degradative machinery requires a set of proteins which are similar in structure and function across most organisms In most of these processes, certain invariant features are observed, including the formation of small non coding RNAs like small interfering RNA (siRNA), microRNA (miRNA) from the dsRNA that initiates the process The systemic transmission and amplification of the silencing signal from its site of initiation.

6. si RNA and mi RNA s i RNA, m i RNA are non-coding RNA s that are associated with RNA interference s i RNA (or short/small interfering RNA) are produced either from exogenous (provided from the outside) ds RNA introduced into the cell in the form of synthetic constructs/viral DNA. It can also be generated endogenously m i RNA ( micro RNA) are always produced from endogenous gene products (coded by the genome) As much as 5% of the human genome is dedicated to encoding and producing the >1,000 miRNAs that regulate at least 30% of our genes       

7. DICER Dicer is the enzyme that cleaves dsRNA into siRNA It belongs to the rnase III nuclease family Rnase III family members are among the few nucleases that show specificity for dsRNAs Dicer has four distinct domains: An amino-terminal helicase domain, Dual RNAse III motifs, A dsRNA binding domain, and A PAZ domain (a 110-amino-acid domain present in proteins like piwi , argo , and zwille /pinhead) Cleavage by dicer is thought to be catalyzed by its tandem rnase III domains .

8. RNA INDUCED SILENCING COMPLEX(RISC) Is a multicomponent protein complex One of the protein components of this complex was identified as a member of the argonaute family of proteins and was termed argonaute2 (AGO2) AGO2 is a 130-kda protein containing polyglutamine residues, PAZ, and PIWI domains characteristic of members of the argonaute gene family AGO2 bears catalytic activity and can cleave the target mRNA RISC also contains other proteins including RNA binding proteins, helicase proteins etc A few molecules of dsRNA are sufficient to degrade a continuously transcribed target mRNA for a long period of time.

9. si RNA mediated RNA- i Initiation: processing of dsRNA into siRNAs Input dsRNA introduced directly or via a transgene or virus is digested into 21-23 nucleotide small interfering RNA s (si RNA s) s iRNAs are formed and accumulate as double-stranded RNA molecules of defined chemical structures Each strand of siRNA has 5-phosphate and 3-hydroxyl termini and 2- to 3-nucleotide 3 overhangs. SiRNAs are produced when the enzyme dicer, a member of the RNase iii family of dsRNA-specific ribonucleases, cleaves dsRNA  in an atp -dependent manner

10. Effector step: The si RNA is then loaded on to a protein complex called as RNA induced silencing complex(RISC). The loading is assisted by the risc -loading complex (RLC) comprised of the DICER and dsRNA-binding protein Inside the RISC the siRNA unwinds in an ATP dependant activity. One of the strands is called the guide strand (the strand complementary to the cognate mRNA) and the other is called the passenger strand. The passenger strand is discarded The guide strand remains associated with the argonaut protein of the RISC The guide strand targets the RISC to the cognate mRNA and the RISC proteins through diverse mechanisms prevent the mRNA from being translated