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.