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MicroRNAs and Other Tiny Endogenous RNAs in C. elegans

MicroRNAs and Other Tiny Endogenous RNAs in C. elegans. Annie Chiang 03.07.03 JClub Ambros et al. Curr Biol 13:807-818. What are microRNAs?. Small (~22nt), non-coding RNAs Excised out of ~70-80nt dsRNA stem-loop precursor miRNA (by Dicer - RNase)

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MicroRNAs and Other Tiny Endogenous RNAs in C. elegans

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  1. MicroRNAs and Other Tiny Endogenous RNAs in C. elegans Annie Chiang 03.07.03 JClub Ambros et al. Curr Biol 13:807-818

  2. What are microRNAs? • Small (~22nt), non-coding RNAs • Excised out of ~70-80nt dsRNA stem-loop precursor miRNA (by Dicer - RNase) • Translational repressor or trigger transcript degradation

  3. microRNA characteristics • ~22nt transcript by Northern blot • Fold-back precursor – 60-80nt • Fold-back precursor w/ lowest free energy • 16/22nt from precursor form miR • No large internal loops/bulges • Phlyogenetic conservation • Increased precursor with reduced Dicer Ambros et al. (2003) RNA 9:277-279

  4. Central dogma

  5. Recently discovered (’93) 58 known miRs in C. elegans Regulatory roles: translation repression transcript degradation Involved in: developmental timing cellular proliferation Apoptosis fat metabolism stress response “switchboard operators” Ambros (2003) Cell 113:673-676 Grosshans and Slack (2002) J Cell Biol 156:17-21 What’s so special about miRs?

  6. Objective: experimentally & computationally detect novel miRs in C. elegans

  7. cDNA cloning

  8. Computational approaches • Comparative genomics (6985 pairs) • Noncoding regions bet C. elegans & C. briggsae (>19nt identity) • 288 candidate pairs (>6.5 hairpin score), Northern • 6 new miRs • Pattern search to known miRs • Patscan – 22nt blocks (10m,1i,0d) or (10m,0d,1i) • Hairpin score, Northern • 3 new miRs

  9. Computational prediction

  10. patscan • Pattern search program • Hairpin loop: stem=20-30bp, loop=3-8bp • p1 = 20...30 3...8 ~p1. • Hairpin loop w/ mismatches (m,i,d) • p1 = 20...30 3...8 ~p1[2,0,0]. • 6 tandem repeat of 4-mers • p1 = 4...4 p1[1,0,0] p1[1,0,0] p1[1,0,0] p1[1,0,0] p1[1,0,0]

  11. Of miRNAs & tncRNAs • 21 novel miRs • cDNA cloning  12 • Comparative genomics  6 • Patscan  3 • Estimated 100-120 miRs total in C. elegans • 33 distinct tncRNAs • Non-coding • Detectable ~22nt by Northern • Lack secondary structure • Not conserved outside C. elegans • Involved in translation repression (not RNAi)

  12. miR families • Conservation indicate common target, reinforced regulation

  13. Developmental and temporal regulation

  14. Antisense cDNAs • 746 seqs -> 551 diff genes • RNAi widespread in worms • X cluster (41seqs) – no known cDNA

  15. Questions?

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