Post on 20-Jan-2016
transcript
StefanAigner
ChristianCarson
Rusty Gage
Gene YeoCrick-Jacobs Center
Salk Institute
Analysis of Small RNAs in Stem Cell Differentiation
Neuronal specification
Neural progenitor(NP)
Embryonic stem(ES)
Neuron (Ne)
Oct4 DAPI
ESNestin Sox1 DAPI
NPMap2a+b Tuj1 DAPI
NE
Cloning of 18-30 nt small RNAs
18 nt24 nt30 nt
SybrGold stained 15% acrylamide/urea gel
ES NP
Adapter ligation
RNA
Reverse transcription,PCR
dsDNA
Cloning and Sequencing
Mapped to repeat-masked human genome (>18bp)
ES 1.04M NP 2.46M Ne 1.83M
2% 2% 4% 2%2%2%
2%3%
74%
7%miRNAWGtRNArRNAHysnRNApiRNAliterature-curatedrepetitive elementsmisc
~4M reads clustered into ~76,000 non-redundant clusters
81% of known mature miRNAs detected
~16K reads per pre-miRNA on average
50% (Novel, genic)
20% (Novel,
intergenic)
30% (non-coding RNAs)
30%
mir-302d
6323reads
Sequence-level resolution
Classifier for novel hairpins (MIRESQUE)
315 of 325 (96%) known microRNA hairpins can be detected reliably
215 MIRESQUE predicted miRNA hairpins (out of 2688 unlabeled samples)
chrX:113772628_3p chr15:81221813_5p chr22:18448149_5p chr19:58867025_5p
35
30
24
18
ES NP ES NP ES NP ES NPnt 5S rRNA
ES NP
let-7c
Summary of miR predictions
0
100
200
300
400
500
600
700
800
900
knownmature mirs
knownmatures in
knownhairpinsdetected
novelmatures in
knownhairpins
matures innovel hairpins
Number
80% of known
430
848 674
113
High resolution allows us to correct annotations
hsa-mir-149 139 363 hsa-mir-149*hsa-mir-522* 24 20 hsa-mir-522hsa-mir-550 6.33 28.33 hsa-mir-550*hsa-mir-302a* 72888 7149.5 hsa-mir-302ahsa-mir-20b 1116.5 2490 hsa-mir-20b*hsa-mir-431 77 390 hsa-mir-431*hsa-mir-136 583.83 706 hsa-mir-136*hsa-mir-551b* 66 27 hsa-mir-551bhsa-mir-377* 277 135.5 hsa-mir-377hsa-mir-30c 261 602 hsa-mir-30c-2*hsa-mir-154 396.5 1062.5 hsa-mir-154*hsa-mir-374a 2754.5 3969.33 hsa-mir-374a*hsa-mir-518e* 102 11.33 hsa-mir-519ahsa-mir-380* 156 130 hsa-mir-380hsa-mir-488* 485 18 hsa-mir-488hsa-mir-30e 765.5 1626 hsa-mir-30e*hsa-mir-500 11 672.5 hsa-mir-500*hsa-mir-550 5.33 28.33 hsa-mir-550*hsa-mir-424 195.5 1084 hsa-mir-424*hsa-mir-144* 20 3 hsa-mir-144hsa-mir-493* 1533.5 222 hsa-mir-493hsa-mir-541* 458 10.5 hsa-mir-541hsa-mir-625 208 210 hsa-mir-625*hsa-mir-505* 1969.5 310 hsa-mir-505
5p 3p‘passenger’‘guide’
24 “Guide” vs “passenger” strand changes ‘target’ predictions.
Correcting 5’ ends of microRNAs
49 known miRNAs have incorrectly annotated 5’ endsMir-34b GTGGTGGTTAcaatcactaactccactgccatCAAAACAAGGEdges ..........>>>------------------<<<<.......+ES AATCACTAACTCCACTGCCAT 1+ES AATCACTAACTCCACTGCCATC 2+ES ATCACTAACTCCACTGCCATCA 2+NP CAATCACTAACTCCACTGCCATCAA 1+NP CAATCACTAACTCCACTGCCAT 1+NP ATCACTAACTCCACTGCCATCA 1.5+NE AATCACTAACTCCACTGCCATC 62
Mir-34b*TAAGAAAAGAtcgtgcatccctttagagtgttACTGTTTGAGEdges ........>>>->--------------<<<<<..........+ES GATCGTGCATCCCTTTAGAGTGT 0.5+ES ATCGTGCATCCCTTTAGAGTG 18+ES ATCGTGCATCCCTTTAGAGTGTT 2.5+ES ATCGTGCATCCCTTTAGAGTGT 264+ES TCGTGCATCCCTTTAGAGTGT 14+ES TCGTGCATCCCTTTAGAGTGTT 0.5+ES TCGTGCATCCCTTTAGAGTG 2+ES TCGTGCATCCCTTTAGAGT 2.5
Sense/antisense microRNAs
Known example:mir-338 11
annotated mirs have s/as pairs
ES
NP
NE
Switching strand preferences
ES
NP
NE
13 microRNAs with strand switches
Khvorova et al, Cell 2003Schwarz et al, Cell 2003
C/D and H/ACA snoRNAs processed as miRNAs?
ES
NP
NE
-8
-6
-4
-2
0
2
4
6
-8 -6 -4 -2 0 2 4 6 8
NP/ES log10(chi-sq)
NE/NP log10(chi-sq)
let-7c, 5p
let-7f-1
mir-221,3p
mir-222, 3p
mir-302a, 5p
Novel, chr10, 3p
mir-103-1
Novel,chr19,5pmir-191, 5p
let-7f-2, 5plet-7g, 5p
mir-107, 3p
mir-423, 5p
mir-92b, 3p
let-7b, 5plet-7e, 5p
let-7a-1, 5p
let-7d, 5p
mir-181a-2, 5pmir-181a-1, 5p
mir-128a, 3p
mir-92a-2, 3p mir-432, 5p
mir-185, 5p
mir-127, 3pmir-29a, 3p
let-7i,5p
mir-99b,5pmir-340,5pmir-140,3p
mir-9-1,5p
mir-128b,3p
mir-124-3,3p
mir-302a, 3p
mir-9-1,3p
Digital expression reveals hES/hNP-specific miRs
ES NENP
ES NENPES NENP
ES NENP
NPES
NE
NP
Small RNA summary
• ES, NP, NE express an unexpectedly complex set of small non-coding RNAs whose biological significance remains to be investigated.
• MIRESQUE designed: We discovered hundreds of novel bona fide microRNAs, dozens of which are in coding regions of genes.
•High resolution allows correct guide/passenger annotations, correct 5’ ends of miRs, •Sense/antisense microRNAs were identified
•MicroRNAs that switch strands
•The microRNA profiles of ES, NP, NE are distinct.
•Novel ES-specific miRs.