miRNA & siRNA

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miRNA & siRNA

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miRNA & siRNA

  1. 1. Origins and Mechanisms of miRNAs and siRNAs Present by: Mozhdeh Mirahadi 1
  2. 2. RNAi Overview During RNAi Double-stranded RNAs cut into short double-stranded RNAs, s(small) i(interfering) RNA's, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi 2
  3. 3. Outlines • IInnttrroodduuccttiioonn • RNA silencing • Definition of RNA interference • MMeecchhaanniissmm ooff RRNNAA iinntteerrffeerreennccee • Argonaute • siRNAs; Sources of siRNA Precursors • RISC • Posttranscriptional Silencing by siRNAs • MicroRNAs • MicroRNA Biogenesis • Posttranscriptional Repression by miRNAs • CCoonncclluussiioonn 3
  4. 4. Introduction RNA i ( RNA Interference ) 4
  5. 5. Definition RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons. 5
  6. 6. Need for interference Defense Mechanism Defense against Infection by viruses, etc As a defense mechanism to protect against transposons and other insertional elements Genome Wide Regulation RNAi plays a role in regulating development and genome maintenance. 30% of human genome regulated 6
  7. 7. RNAi: Silencing in Cenorhabditis elegans dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function. 7
  8. 8. Cenorhabditis elegans RNAi can be induced in C. elegans in three simple ways: Injection of dsRNA into the worm gonads Soaking the worms in dsRNA solution Feeding the worms engineered bacteria producing dsRNA 8
  9. 9. Mechanism of RNAi RNA i ( RNA Interference ) 9
  10. 10. 10
  11. 11. The Players In Interference RNA siRNA: dsRNA 21-22 nt. miRNA: ssRNA 19-25nt. Encoded by non protein coding genome RISC: RNA induced Silencing Complex, that cleaves mRNA Enzymes Dicer : produces 20-21 nt cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre miRNA; which is later processed by Dicer 11
  12. 12. siRNAs • Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing • RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation • A single base pair difference between the siRNA template and the target mRNA is enough to block the process. • Each strand of siRNA has: • a. 5’-phosphate termini • b. 3’-hydroxyl termini • c. 2/3-nucleotide 3’ overhangs 12
  13. 13. siRNA design 21-23nt 2-nt 3' overhangs ( UU overhangs ) G/C content: 30-50%. No basepair mismatch Synthesised siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). BLAST : eliminate any target sequences with significant homology to other coding sequences. 13
  14. 14. Generation of small interference RNA 14
  15. 15. miRNA Originate from capped & polyadenylated full length precursors (pri-miRNA) Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA) 15
  16. 16. 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. And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre-transcriptionally (RNA induced transcriptional silencing or RRIITTSS) 16
  17. 17. Dicer RNase III-like dsRNA-specific ribonuclease Enzyme involved in the initiation of RNAi. It is able to digest dsRNA into uniformly sized small RNAs (siRNA) Dicer family proteins are ATP-dependent nucleases. Rnase III enzyme acts as a dimer Loss of dicer→loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer is a conserved protein) 17
  18. 18. RISC RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA Unwinding of double-stranded siRNA by ATP independent helicase. The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand. 18
  19. 19. Summary of Players Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) Drosha and Dicer are RNase III enzymes Pasha is a dsRNA binding protein Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran and GTP Argonautes are RNase H enzymes 19
  20. 20. Mechanism of RNA interference 20
  21. 21. Mechanism of RNA interference 21
  22. 22. Over Figure 1. Core Features ooff mmiiRRNNAA aanndd ssiiRRNNAA SSiilleenncciinngg 22
  23. 23. Argonaute: At the Core of RNA Silencing The Argonaute superfamily can be divided into three separate subgroups: the Piwi clade that binds piRNAs, the Ago clade that associates with miRNAs and siRNAs, third clade that has only been described thus far in nematodes. 23
  24. 24. Over Figure 2. A Diversity ooff ssiiRRNNAA SSoouurrcceess 24
  25. 25. RISC Assembly and siRNA Strand Selection Although single-stranded siRNAs can load directly into purified Argonaute proteins, the double-stranded siRNAs that are generated by Dicer cannot and rely instead upon siRISC assembly pathways (Figure 2). 25
  26. 26. Over Figure 3. Mechanisms of siRNA Silencing 26
  27. 27. Amplification of siRNA 27
  28. 28. siRNAs Can Induce Heterochromatin Formation siRNAs are not restricted to posttranscriptional modes of repression. In 2002, siRNAs were shown to induce heterochromatin formation in S. pombe, consistent with earlier reports of transcriptional gene silencing (TGS) in plants. 28
  29. 29. Illustration of miRNA processing 29
  30. 30. Another View 30
  31. 31. MicroRNA Biogenesis MicroRNAs in the plant and animal (Figure 4) 31
  32. 32. Over Figure 4. Biogenesis of miRNAs and Assembly into miRISC in Plants and Animals 32
  33. 33. MicroRNA Associations miRNA strand miRNA* strand In Drosophila in humans, C. elegans, and Drosophila indicates 33
  34. 34. Posttranscriptional Repression by miRNAs The miRNA acts as an adaptor (Figure 5) The degree of miRNA-mRNA complementarity has been considered a key determinant of the regulatory mechanism. 34
  35. 35. Over Figure 5. Possible Mechanisms of miRISC-Mediated Repression 35
  36. 36. Conclusions dsRNA needs to be directed against an exon, not an intron in order to be effective Homology of the dsRNA and the target gene/mRNA is required Targeted mRNA is lost (degraded) after RNAi The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism) ssRNA does not work as well as dsRNA 36
  37. 37. Thank you! Any guestion? 37

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