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Mechanisms for RNAi

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Mechanisms for RNAi

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    1. Mechanisms for RNAi /miRNA pathways Cell 107, 309-321 (2001) Cell 115, 199-208 (2003) Science 306, 1377-1380 (2004) Cell 123, 607-620 (2005) Cell 130, 287-297 (2007) Cell 130, 299-308 (2007)

    4. Role for a ribonuclease (Dicer) in the initiation step of RNAi RNAi is a multi-step process that begins with ATP-dependent processing of long dsRNA into 21-23 nt siRNA by Dicer Dicer, a RNase III protein. RNase III enzymes act as dimers to cleave both strands of dsRNA, leaving two-nucleotide 3’ overhanging ends. Dicer contains: 1. An ATP-dependent RNA helicase domain. 2. A Piwi/Argonaute/Zwille (PAZ) domain. 3. A dsRNA-binding domain. 4. Two RNase III domains

    6. Role of ATP in the RNAi pathway

    7. Native siRNA can be isolated by gel filtration

    8. Column fraction for RNAi activity indicate siRNA are true intermediates in RNAi

    9. Interference mediated by siRNA also requires ATP

    10. Target recognition and cleavage are ATP-independent

    11. The second ATP-dependent step lies downstream of dsRNA processing but upstream of target recognition

    13. Analysis of siRNP complex formation by gel filtration

    14. The majority of siRNAs associate with protein are present in a ~360 kDa siRNP complex that is not competent to cleave a target RNA, whereas a minority of the siRNAs are in a smaller, highly active complex.

    15. Unwinding of siRNA during the RNAi reaction is ATP-dependent

    16. The siRNA is almost entirely double-stranded in the inactive ~360kDa siRNP complex.

    17. 5’phosphates are required for siRNP formation

    19. RNA-induced silencing complex (RISC) A four sequential steps pathway for RNAi ATP-dependent processing of dsRNA into small interfering RNAs (siRNA). Incorporation of siRNA into an inactive protein/RNA complex (siRNP). ATP-dependent unwinding of siRNA duplex to generate an active complex (RISC). ATP-independent recognition and cleavage of the RNA target.

    20. siRNA strand-selection and incoporation during RISC assembly

    22. Functionally asymmetric siRNA duplexes.

    23. The two siRNA strands are equally effective as single strands but show dramatically different activities when paired to each other This suggest the asymmetry in their function is established at a step in the RNAi pathway before the encounter of the programmed RISC with its RNA target.

    24. The two strands of an siRNA duplex do not equally populate into the RISC.

    25. Thermodynamic bias ?!

    26. 5’ terminal, single-nucleotide mismatched makes siRNA duplexes functionally asymmetric.

    27. 5’ terminal, single-nucleotide mismatched makes siRNA duplexes functionally asymmetric.

    28. A single hydrogen bond can determine which siRNA strand directs RNAi

    29. Internal stability of dsRNA is (composition) sequence-dependent

    30. The first four base pairs of the siRNA duplex determine strand-specific activity.

    31. Enhanced flexibility at the 5’-AS terminal position and the low internal energy across the duplex (especially at the region 9-14) are strongly correlated with siRNA function.

    33. Implication of siRNA asymmetry for miRNA biogenesis

    34. The difference in the base pairing of the first five nucleotides of the miRNA versus miRNA* strand can predict which strand to accumulate in vivo.

    35. RISC-loading complex (RLC) containing ds siRNA, R2D2 and Dcr2 can initiate siRNA unwinding

    36. The RLC can sense siRNA thermodynamic asymmetry, thereby determining which strand enters the RISC

    37. DCR almost always near the 5’ end of the guide strand and R2D2 near the 5’ end of the passenger strand

    38. R2D2 as asymmetry sensor for two aspects of siRNA structure: the stability of its 5’ end and the presence of a 5’ phosphate group.

    39. Binding of Ago2 facilitates the release R2D2 from siRNA.

    41. Passenger-strand cleavage

    42. Cleavage is restricted to the passenger strand of the siRNA duplex

    43. Passenger strand cleavage require the core component of the RLC, Dcr-2 and R2D2 in addition to the Ago2

    45. SiRNA can function as miRNA The complementarity determines the pathway to mRNA cleavage or translation repression.

    46. miRNA-diected cleavage

    47. The sorting of siRNA/miRNA among Agos Cell 130, 287-297 (2007)

    48. The two alternative model for sorting

    49. Components of both pathways are required to silence a reporter with perfect matches to miR-277

    50. Only components of the miRNA pathway are required for imprefectly matched target

    51. Most endogenous miR-277 is not associated with Ago1

    52. Repression via perfect matched or imperfect match dependent differently on Dcr2/R2D2 loading activity

    53. Ago1 is a poor endonuclease

    55. siRNAs sorting in Fly Cell 130, 299-308 (2007)

    56. RNA duplex structure determines the partition

    58. Dcr-2/R2D2 affect competition between Ago1-RISC and Ago2-RISC assembly

    60. Ago2-RISC activity coincides with C1 complex and a central mismatch impairs C1 formation

    62. The Ago1-loading pathway selects small RNAs with central mismatches.

    63. Only let-7/let-7* duplex efficiently assembles mature Ago1-RISC

    64. The structure generated determine the partition between Ago1- and Ago2-RISC

    65. Model for small silencing RNA sorting in Fly

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