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RNA Interferrance

CMB-234:Molecular Biology. RNA Interferrance. Atta ur Rahman school of biological Science National University of Sciences & Technology. RNA Interference.

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RNA Interferrance

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  1. CMB-234:Molecular Biology RNA Interferrance Atta ur Rahman school of biological Science National University of Sciences & Technology

  2. RNA Interference • RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules •  In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans, which they published in 1998

  3. Interference can be at • Transcriptional gene silencing (TGS) • Post transcriptional gene silencing (PTGS) • Co - transcriptional gene silencing (CTGS) • Chromatin dependent gene silencing (CDGS)

  4. Cont’d • Three types include • Micro RNA (21 – 25nt) • Small interfering RNA (21 – 25nt) • PIWI interacting RNA (24 – 31nt)

  5. Generation of RNAi

  6. siRNA Aberrant RNA 3’ 5’ Convergent promoters Dicer Dicer TRAMP RDRP Degradation of RNA by forming exosome

  7. miRNA DROSHA DICER

  8. piRNA AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA PIWI AUB or AGO- 3

  9. RNA Silencing Pathways

  10. siRNA Argonaut : - PIWI - PAZ - MID Dicer = a RNase - III This process can take place here as well RISC = RITS RISC 20 - 25 nt long NUCLEUS CELL

  11. miRNA Drosha & Dicer Argonaut : - PIWI - PAZ - MID May be present in here RISC ssRNA ~20 - 25 nt NUCLEUS Translational inhibition CELL

  12. piRNA Mechanism of action of pi RNA is not yet fully elucidated but it is known that it involves the slicer activity of PIWI Clade It is responsible for controlling the movement of transposons in the genome NUCLEUS CELL

  13. Biological Functions

  14. Linking heterochromatin to RNAi

  15. Cont… • Gene regulation • RNA sequences (siRNA and miRNA) that are complementary to parts of a promoter can increase gene transcription, a phenomenon dubbed RNA activation. • Part of the mechanism for how these RNA upregulate genes is known: dicer and argonaute are involved, possibly via histone demethylation. • miRNAs have also been proposed to upregulate their target genes upon cell cycle arrest, although the underlying mechanisms have not been elucidated.

  16. Regulation of gene expression Maintenance of genome stability Concentrated in centromeres in all except yeast Important role in chromosome transmission Also in telomeres & genome loci Repetitive DNA sequences & Transposons HETEROCHROMATIN Arrangement 1. Protein & RNA - Sequence 2. Changes in chromatin structure x H3K9 methylation done by Chromodomain-1(Chp1) x loss of Chp-1, decrease in methylation x purification of Chp-1 has shown its association with Ago1 in RITS x This means that RNAi may have some role in heterochromatin silencing as it activates the same proteins that are activated by other cellular factors for this control

  17. RNAi in DNA & Chromatin regulation

  18. Cont… • Gene Down regulation • Endogenously expressed miRNAs, are most important in translational repression and in the regulation of development, especially on the timing of morphogenesis and the maintenance of undifferentiated or incompletely differentiated cell types such as stem cells

  19. Cont… RNAi IS REQUIRED FOR SOME BUT NOT ALL CHROMATIN MEDIATED SILENCING EVENTS • In Fission yeast & some ciliates, loss of heterochromatin gene silencing by del in • Argonaute (Ago1) • Dicer (dcr1) • RNA dependent RNA polymerase (rdp1) • In Arabidopsisthaliana, RNA directed DNA methylation (H3K9)requires 2 proteins • Dicer (DLC-3) • Argonaute (AGO-4) • In Drosophilamelanogaster, repeat induced gene silencing by • Polycomb group of genes (PcG) • PIWI • DCR-1

  20. Immune response • RNA interference is a vital part of the immune response to • Viruses and • Other foreign genetic material, especially in plants where it may also prevent the self-propagation of transposons.

  21. Future Prospects Most mechanisms still unknown In future we can employ this mechanism to up or down regulate a gene, or to fight viral infections

  22. Reference: Small RNAs in transcriptional gene silencing and genome defence Danesh Moazed Nature457, 413-420 Thank you!

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