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RNA-mediated regulation: How many functions does it serve?

RNA-mediated regulation: How many functions does it serve?. BioInformatics Seminar Series Kriton Kalantidis IMBB and Dept of Biology, UoC. Effects of expression of CHS sense and antisense RNA on flower pigmentation in Petunia. van der Krol et al.,.

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RNA-mediated regulation: How many functions does it serve?

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  1. RNA-mediated regulation:How many functions does it serve? BioInformatics Seminar Series Kriton Kalantidis IMBB and Dept of Biology, UoC

  2. Effects of expression of CHS sense and antisense RNA on flower pigmentation in Petunia van der Krol et al., Plant Cell 2:291 (1990) + Plant Mol Biol14:457 (1990).

  3. RNA silencing • RNA mediated impairment of transcription- TGS -methylation (shRNAs, rasiRNAs and siRNAs?) • RNA mediated impairment of translation -PTGS (siRNAs and some miRNAs) and -translational arrest (miRNAs)

  4. specific degradation of target RNA changes in chromatin structure RNA interference (RNAi) regulation of “epigenome” short interfering RNA (siRNA) short heterochromatic RNA (shRNA) small RNA may impair gene expression small RNAs Processed from endogenous transcripts regulation of genes particularly in development micro RNA (miRNA) and more…

  5. The processing pathway of siRNA & miRNA from Dykxhoorn et al., (2003) Nature Reviews 4, 457-466.

  6. 5’ terminal phosphates 5’ P - N N N N N N N N N N N N N N N N N N N N N N - P 5’ N N N N N N N N N N N N N N N N N N N N N - - N N N two 3’ protruding unpaired nucleotides N N N - - N short interfering RNA (siRNA) 3’ 3’ N N ca 21 Nucleotides

  7. RNA mediated regulationKey Players • The Dicers, one or more members 1- S. pombe, C. elegans, Mammals 2- Drosophila 4- Dicots (plants, e.g. Arabidopsis) 5- Monocots (plants e.g. Rice) At least two diversified functions: miRNA processing and siRNA generation

  8. RNA mediated regulationKey Players 2. • Argonaute proteins (Piwi and Paz domains responsible for the slicing of the target molecule), often part of the RISC complex, very large family of genes • RdRP, present only in some organisms (plants, worms, fungi) • Other complexes (Drosha/Pasha, RITS etc)

  9. Biological role of silencing  Protection against invasive RNAs especially RNA viruses  Silencing of transposable elements  Regulation of expression of endogenous genes

  10. MicroRNAs • short (20-25nt) RNA molecules • translated as precursor RNA molecule • post transcriptional gene regulation • target mRNA for cleavage or translational repression • very abundant class of genes

  11. miRNAs are typical RNA-polII transcripts

  12. Open Questions1. RNA silencing • Initiation: (plants, worm, fly) What is aberrant RNA? Is there a threshold? • Maintenance: (plants, worm, fly?) Is there an endogenous “cooling system” “Degradive PCR”? Needs methylation? • Systemic Spread: (plants, worm?) A threshold needed? What is the silencing signal? How does it move?

  13. Integrated Model for RNA silencing Hutvagner and Zamore, Curr. Op. Genet. & Dev. 12, 225-232 (2002)

  14. Open Questions1. RNA silencing • Initiation: (plants, worm, fly) What is aberrant RNA? Is there a threshold? • Maintenance: (plants, worm, fly?) Is there an endogenous “cooling system” “Degradive PCR”? Needs methylation? • Systemic Spread: (plants, worm?) A threshold needed? What is the silencing signal? How does it move?

  15. Open Questions2. miRNAs • How many genes do they regulate? • What is the importance of miRNA families? • What gives miRNAs their expression specificities? • What is the mode of their function, translational arrest, and at what stage?

  16. RNA silencing related questionscurrent answears • Initiation: What is aberrant RNA?- ? Must be qualitative specificity Is there a threshold?- seems to be / We have evidence • Maintenance: “Degradive PCR”? It involves RdRP activity, not proven Is there an endogenous “cooling system”, maybe Eri involved Needs methylation? Most people seem to think so (?) • Systemic Spread: A threshold needed?-seems to/ we have some evidence What is the silencing signal? RNA but of what sort? How does it move? Together with metabolites (we say)

  17. Transgenic GFP expressing N.benthamiana is our system

  18. Multiple thresholds seem to regulate initiation and spread of silencing

  19. Short Range Silencing: A type of silencing that does not spread b a

  20. Short Range Silencing is (likely): • A post transcriptional phenomenon • Caused by small amounts of siRNAs

  21. Local & systemic Short range silencing A simple model for the “red spots”: Inducer of RNAi If below if if Ignore Threshold 2 Threshold 1 below above If above

  22. ERI-1:AnRNasethat deactivates siRNAs 5’ P - N N N N N N 3' N N N N N N N N N N N N N N N N N N N N - P N N N N N N N N N N N N N N N N 3’ N N 5’ ca 21 Nucleotides ERI-1 5’ P - N N 3’ N N N N N N N N N N N N N N N N N N N N N N - P 5’ 3’ N N N N N N N N N N N N N N N N ca 21 Nucleotides

  23. Eri1 was suppressed with the use of hairpin constructs, one using the Arabidopsis and the other the tobacco ortholog • Text SalI (1) NotI (962) RB CaMV 35S KpnI (2362) Eri 600bp fragment SalI (3012) intron Tn7 Spr/Str pART27 antiEri • AccI (4452) 15840bp • Eri 600bp fragment • ocs 3' NotI (6002) nptII SalI (8222)

  24. Analysis of Eri Expression, following agroinfiltration with Eri-hairpin, using RT-PCR Nt Nb 3rd d 6th d 10th d transgenic

  25. Suppression ofEri1 in N. benhtamiana transforms short range silencing to local and systemic

  26. Some Conclusions • The short range signal is likely siRNA and moves faster but not solely through cytoplasmic canals • SRS can be induced by silencing inducers at concentrations below an arbitrary threshold. • Suppression of ERI causes the transition from short range to systemic silencing

  27. The silencing signal moves from metabolic source to metabolic sink

  28. Systemic silencing can happen spontaneously or can be induced Spontaneous systemic silencing Induced systemic silencing

  29. Grafting experiments to monitor PTGS spread transgene transgene Non silenced silenced

  30. Grafting experiments to monitor PTGS spread transgene transgene Non-silenced silenced

  31. Silencing spreads from root-stock to scion Transgene Transgene silenced Non-silenced

  32. Systemic spread,The questions addressed: • What determines the movement of the silencing signal? • What influences the efficiency of silencing transmission?

  33. Silencing does not necessarily move from stock to scion

  34. 32Pi can be used to track the flow of phloem sap

  35. The silencing signal recapitulates the pattern of phloem flow

  36. Conclusions? • The silencing signal seems to follow the phloem flow • The plant architecture influences the movement of the signal • There may be a threshold of signal that needs to be exceeded to establish silencing

  37. Current answers on miRNAs • How many –and which- genes do they regulate? – hundreds to thousands (estimated up to 20% of the total) found some • What is the importance of miRNA families? Multiple levels of regulation? • What gives miRNAs their expression specificities? polII promoters, any specificities? • What is the mode of their function, translational arrest, and at what stage? Both arrest and degradation have been found

  38. Identifying miRNA regulated genes related to cancer

  39. Various ways of identifying miRNA targets: • Bioinformatics • Experimental In both cases the interaction has to be experimentally validated!

  40. The Microinspector program A software to detect miRNA binding sites in a given mRNA target A service forthose interested in finding whether a particular gene has miRNA binding sites currently accesible at:www.imbb.forth.gr/ webtools.html

  41. miRNAs and oncogenes We got all the human UTR region’s sequences (http://bighost.area.ba.cnr.it/BIG/UTRHome/) and extracted all the oncogenes’ UTRs from them

  42. miRNAs and oncogenes • We ran the dataset with the modified version of microInspector which can grab a whole set of sequences and analyze them for all miRNAs in a particular organism (in this case – Human miRNAs) • From the results we selected manually only the best structures (binding sites) - about 60 onco and onco-related genes.

  43. miRNAs and oncogenes Example - 6 results from the result table around Bcl2/miR16 row

  44. Johnson et al. 2005. RAS is regulated by the let-7 micro RNA family; Cell, 120(5):635-47. • Cimino A. et al. 2005. miR15 and 16 induce apoptosis by targeting Bcl-2. PNAS Sept15.

  45. miRNAs and oncogenes Candidate genes Additional gene ► Bcl2, oncogene,anti-apoptotic, correlation with CLL, possible regulation by mir16 found with microInspector

  46. Binding of miRNA 16 and 15 to Bcl-2 and MYCBD respectively miR15/MYCBP miR16/Bcl-2

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