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. What’s so special about miRs? 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

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?