Food processing presentation for bsc agriculture hons
RNA interference
1. RNA INTERFERENCE siRNA and miRNA Ms.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. Timeline short RNAs identified in plants RNAi shown in vitro siRNAs identified Dicer identified genome-wide RNAi screens begin RNAi used against HIV 1990 cosuppression of purple color in plants dsRNA injection in worms 1998 1999 RISC activity partially purified 2000 2001 2002
4. RNA Interference Phenomena first observed in petunia Attempted 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
6. RNAi Mechanism The 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 enzyme The PAZ domain, a module that binds the end of dsRNA, is directly connected to the RNase IIIa domain by a long alpha helix Dicer 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”.
11. Mechanism of RNAi The 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 RNAi The 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 levels important for development and differentiation second miRNA (let-7) discovered in 2000
14. micro RNA (miRNA) A miRNA is a ssRNA of ~22 nucleotides in length Generated 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
19. miRNA MECHANISM RNase 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,
24. miRNAs as cancer genes Overexpression 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 life Brain 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)
28. RNAi, a new therapeutic strategy against viral infection Prevents viral infection Inhibits the expression of viral antigens Suppresses the transcription of viral genome Blocks viral replication Silences viral accessory genes Hinders the assembly of viral particles & Displays roles in virus-host interactions
30. Computational Methods for identification of siRNA siRNA targeted sequence is usually 21 nt in length. Avoid regions within 50-100 bp of the start codon and the termination codon Avoid 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 group Select 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)YNN Target 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 siRNAs GenScript - siRNA Design IDT SciTools RNAi Design Ambion scientists find that ~50% of siRNAs designed using this tool will reduce target gene expression by >50%.
39. Rules used in SVM RNAi GC 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.