micrornas and other tiny endogenous rnas in c. elegans annie chiang 03.07.03 jclub ambros et al....
TRANSCRIPT
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) Translational repressor or trigger transcript
degradation
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
Central dogma
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
Objective: experimentally & computationally detect novel miRs in C. elegans
cDNA cloning
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
Computational prediction
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]
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)
miR families
Conservation indicate common target, reinforced regulation
Developmental and temporal regulation
Antisense cDNAs
746 seqs -> 551 diff genes RNAi widespread in worms X cluster (41seqs) – no known cDNA
Questions?