RNA interference
- 1. RNA INTERFERENCEsiRNA and miRNAMs.ruchiyadavlectureramity institute of biotechnologyamity universitylucknow(up)
- 2. RNA interference (RNAi)RNA interference (RNAi) is a highly evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homogolous mRNA sequences. It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegansand later found in a wide variety of organisms, including mammals
- 3. Timelineshort RNAs identified in plantsRNAi shown in vitrosiRNAs identifiedDicer identifiedgenome-wide RNAi screens beginRNAi used against HIV1990cosuppression of purple color in plantsdsRNA injection in worms19981999RISC activity partially purified200020012002
- 4. RNA InterferencePhenomena first observed in petuniaAttempted to overexpress chalone synthase (anthrocyanin pigment gene) in petunia. (trying to darken flower color) Caused the loss of pigment.Called co-suppression because suppressedexpression of both endogenous gene and transgene
- 5. RNA Interference ApproachesFour types of responses induced by dsRNA
- 6. RNAi MechanismThe RNAi pathway can be divided into three major steps: First is the conversion of dsRNA input into 21-23bp small fragments by the enzyme Dicer; Secondly the loading of small RNAs into large multiprotein complex RISC Lastly the sequence specific silencing of the cognate gene by RISC that is guided by the small RNA fragment.
- 7. SMALL INTERFERING RNA(siRNA)dsRNAs are cleaved into 21-23 nt segments (“small interfering RNAs”, or siRNAs) by an enzyme called Dicer
- 8. Structure of Dicer enzymeThe PAZ domain, a module that binds the end of dsRNA, is directly connected to the RNase IIIa domain by a long alpha helixDicer enzyme plays two biochemically distinct roles in the RNAi mechanics. It functions to generate siRNA molecules Role in loading one of the two siRNA strands onto RISC complex
- 9. RNA-inducing silencing complex (risc)The siRNAs are incorporated into the (RISC) which consists of an Argonaute (Ago) protein as one of its main components. The Argonaute protein is considered as the catalytic engine or the signature component of the RISC Ago cleaves and discards the passenger (sense) strand of the siRNA duplex leading to activation of the RISC.Ago cuts mRNA targets guided by siRNA via its endonuclease nicknamed “slicer”.
- 10. The Mechanism of RNAi
- 11. Mechanism of RNAiThe silencing mechanism of RNAinterference involves two steps.Initiation step: - first the dsRNA get processed into 21-23 nucleotides small interfering RNAs (siRNAs), which have also been called “guide RNAs, by an RNase III like enzyme called Dicer.Effecter step: - Then, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs),unwinding in the process. An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC
- 12. Mechanism of RNAiThe active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA ~12 nucleotides from the 3' terminus of the siRNA and destroys the cognate RNA.
- 13. micro RNA (miRNA)first discovered in 1993 by Victor Ambros in C. elegans:lin-4 RNA regulates LIN-14 protein levels, but not lin-14 mRNA levelsimportant for development and differentiationsecond miRNA (let-7) discovered in 2000
- 14. micro RNA (miRNA)A miRNA is a ssRNA of ~22 nucleotides in lengthGenerated by the RNase-III-type enzymes Drosha and Dicer from an endogenous transcript that contains a local hairpin structure.pri-miRNAs contain cap and poly(A) tail and are transcribed by RNA Polymerase II
- 15. micro RNA (miRNA)Gene expression regulation
- 16. Created by similar process to siRNA
- 17. Generally prevents binding of ribosomeMicroRNA’sMicroRNA’s (miRNA’s) are small noncoding RNA molecules that regulate eukaryotic gene expression at the translation levelRISC = RNA-induced Silencing Complex
- 18. MicroRNA(miRNA)
- 19. miRNA MECHANISMRNase III enzyme Drosha, which cleaves the stem ~22 nt away from the terminal loop to generate an ~65-nt pre-miRNA hairpin intermediate .Drosha leaves a characteristics 2-nt 3` overhang The pre-miRNA is transported to the cytoplasm by Exportin-5,where it interacts with a second RNase III enzyme called Dicer.Dicer binds the 2-nt 3` overhang found at the base of the pre-miRNA hairpin and cleaves ~22nt away from the base, removing loop & leaving another 2-nt 3` overhang .The resultant duplex intermediate interacts with RISC components,
- 21. miRNA MECHANISMAgo 2 binding of siRNA and passenger strand cleavage.
- 22. miRNA vs. siRNA pathways
- 23. miRNAs as cancer genes
- 24. miRNAs as cancer genesOverexpression of miRNAs—for instance, by amplification of the miRNA-encoding locus—could decrease expression of the target, such as a tumor suppressor gene. Underexpression of miRNAs—for instance, by deletion or methylation of the miRNA locus—could result in increased expression of a target such as an oncogene.
- 25. miRNAs play important roles in all aspects of lifeBrain development (miR-430)Patterning of nervous system (miR-273)Pancreatic islet-cell development (miR-375)Adipocyte differentiation (miR-143)Limb patterning (miR-196)Heart development (miR-1)Programmed cell death (miR-14)
- 26. Genomic organization of miRNAs
- 27. INTEGRATION OF RNAi IN DRUG DISCOVERY
- 28. RNAi, a new therapeutic strategy against viral infectionPrevents viral infectionInhibits the expression of viral antigensSuppresses the transcription of viral genomeBlocks viral replicationSilences viral accessory genes Hinders the assembly of viral particles & Displays roles in virus-host interactions
- 30. Computational Methods for identification of siRNAsiRNA targeted sequence is usually 21 nt in length. Avoid regions within 50-100 bp of the start codon and the termination codonAvoid stretches of 4 or more bases such as AAAA, CCCC Avoid regions with GC content <30% or > 60%. Avoid repeats and low complex sequence Avoid single nucleotide polymorphism (SNP) sites Perform BLAST homology search to avoid off-target effects on other genes or sequences .Avoid sequences that share a certain degree of homology with other related or unrelated genes
- 31. Tom Tuschl's rules(http://www.rockefeller.edu)The first set of empirical rules for siRNA design was compiled by Tuschl’s groupSelect targeted region from a given cDNA sequence beginning 50-100 nt downstream of start condon First search for 23-nt sequence motif AA (N19). If no suitable sequence is found, then, Search for 23-nt sequence motif NA(N21) and convert the 3' end of the sense siRNA to TT Or search for NAR(N17)YNNTarget sequence should have a GC content of around 50% less than 60%R=Adenine or Guanine (Purines); Y=Thymine or Cytosine (Pyrimidines); N = Any.
- 33. siRNA Validation Tools DEQOR, a web-based tool for the Design and Quality Control of siRNAsGenScript - siRNA DesignIDT SciTools RNAi DesignAmbion scientists find that ~50% of siRNAs designed using this tool will reduce target gene expression by >50%.
- 36. miRNAs
- 39. Rules used in SVM RNAiGC content 30-52% preferred. At least 3 "A/U" at sense position 15-19 preferred. Internal hairpin is penalized. "A" at position 19 is preferred. "A" at position 3 is preferred. "U" at position 10 is preferred. "G/C" at position 19 is penalized. "G" at position 13 is penalized. Consecutive repeats (e.g., AAAA, GGGG) of more than 3 bases are penalized.
- 40. SVM RNAi 3.6
- 41. SVM RNAi 3.6