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?