ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ugucggcucCGGGAGAGCCAg 5' || |||||||| Target 5' attccttttGCACTCTCGGTt 3'	55 - 75	152.00	-16.90
2	miRNA 3' ugUCG--GCUCCGGG----AGAGCCAg 5' ||| :||| ||| |||:||| Target 5' gtAGCAGTGAGACCCCTTGTCTTGGTg 3'	675 - 701	143.00	-19.80
3	miRNA 3' ugucGGCUC-CGG--GAGAGCCAg 5' |:||| |:: ||:||||| Target 5' tttcCTGAGTGTTTGCTTTCGGTt 3'	438 - 461	139.00	-17.90

Mutant ID	Mutant Position	Mutant Source
COSN26967316	3	COSMIC
COSN30501043	22	COSMIC
COSN30101525	23	COSMIC
COSN30506878	24	COSMIC
COSN31600915	45	COSMIC
COSN30188704	73	COSMIC
COSN30159033	92	COSMIC
COSN31564141	148	COSMIC
COSN9608101	170	COSMIC
COSN31596677	267	COSMIC
COSN5678387	597	COSMIC
COSN16051921	665	COSMIC
COSN9259357	693	COSMIC
COSN31521675	956	COSMIC
COSN31591772	1038	COSMIC
COSN15613914	1041	COSMIC
COSN31596201	1276	COSMIC
COSN23821512	1291	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1174968187	1	dbSNP
rs141302257	3	dbSNP
rs1283865676	8	dbSNP
rs774744191	11	dbSNP
rs374051764	13	dbSNP
rs1230879040	15	dbSNP
rs746411083	19	dbSNP
rs1296306545	21	dbSNP
rs772655534	22	dbSNP
rs372860051	23	dbSNP
rs761328130	24	dbSNP
rs764715088	25	dbSNP
rs772918732	27	dbSNP
rs139800615	28	dbSNP
rs1171841191	36	dbSNP
rs1251312092	39	dbSNP
rs751396385	41	dbSNP
rs754685764	42	dbSNP
rs767489527	45	dbSNP
rs903764904	46	dbSNP
rs1285441223	47	dbSNP
rs752621698	49	dbSNP
rs1431058881	54	dbSNP
rs557401710	55	dbSNP
rs1000768485	56	dbSNP
rs532531928	57	dbSNP
rs1342715459	61	dbSNP
rs1395142223	65	dbSNP
rs1253029234	67	dbSNP
rs1229655742	69	dbSNP
rs149793493	71	dbSNP
rs1312260758	72	dbSNP
rs1234201167	85	dbSNP
rs889594276	97	dbSNP
rs1008481703	98	dbSNP
rs1008824965	101	dbSNP
rs1019822470	103	dbSNP
rs561303925	109	dbSNP
rs993820769	113	dbSNP
rs1455917166	117	dbSNP
rs11557863	122	dbSNP
rs764229813	122	dbSNP
rs1462441943	144	dbSNP
rs961602139	149	dbSNP
rs540565871	157	dbSNP
rs1409329985	158	dbSNP
rs565524631	159	dbSNP
rs1026641226	165	dbSNP
rs1438917333	168	dbSNP
rs768375671	183	dbSNP
rs990543704	189	dbSNP
rs916330579	191	dbSNP
rs776263675	192	dbSNP
rs761512350	193	dbSNP
rs1233993009	194	dbSNP
rs764873008	199	dbSNP
rs1337156741	201	dbSNP
rs532701600	203	dbSNP
rs530883324	212	dbSNP
rs1336772096	214	dbSNP
rs992323203	220	dbSNP
rs145020321	226	dbSNP
rs917991366	242	dbSNP
rs1462583160	247	dbSNP
rs945489131	251	dbSNP
rs1158018684	256	dbSNP
rs1042472215	259	dbSNP
rs981894475	264	dbSNP
rs180745617	269	dbSNP
rs925280400	270	dbSNP
rs935241227	276	dbSNP
rs1469907004	277	dbSNP
rs936677479	277	dbSNP
rs1307359881	278	dbSNP
rs1049628471	292	dbSNP
rs1205875425	303	dbSNP
rs530499329	305	dbSNP
rs1231554362	311	dbSNP
rs1008053954	316	dbSNP
rs768061257	323	dbSNP
rs1040928236	327	dbSNP
rs1362464376	328	dbSNP
rs773661199	338	dbSNP
rs1053638934	341	dbSNP
rs914540451	353	dbSNP
rs377602196	361	dbSNP
rs1212651307	365	dbSNP
rs552362868	365	dbSNP
rs947394839	372	dbSNP
rs1465382883	381	dbSNP
rs766886448	382	dbSNP
rs1195474498	383	dbSNP
rs183678135	384	dbSNP
rs993892953	387	dbSNP
rs1184194490	389	dbSNP
rs1188015899	392	dbSNP
rs752043681	395	dbSNP
rs1264156587	397	dbSNP
rs1429010233	400	dbSNP
rs889854097	402	dbSNP
rs1026747503	405	dbSNP
rs952377774	425	dbSNP
rs534865898	434	dbSNP
rs1041231732	436	dbSNP
rs1296151811	443	dbSNP
rs546481385	455	dbSNP
rs571478532	458	dbSNP
rs755362166	459	dbSNP
rs1027241819	464	dbSNP
rs1395211121	468	dbSNP
rs149082487	470	dbSNP
rs761060369	482	dbSNP
rs1298428980	484	dbSNP
rs1333145637	486	dbSNP
rs1233116601	490	dbSNP
rs1167361091	497	dbSNP
rs966863965	498	dbSNP
rs1244029837	500	dbSNP
rs1023325735	501	dbSNP
rs73669746	506	dbSNP
rs188397595	511	dbSNP
rs1213481392	516	dbSNP
rs1482332026	518	dbSNP
rs1274591905	520	dbSNP
rs756340594	534	dbSNP
rs1240963057	535	dbSNP
rs925362643	536	dbSNP
rs923742210	538	dbSNP
rs1355705986	540	dbSNP
rs1050224402	542	dbSNP
rs956541862	543	dbSNP
rs1369833823	545	dbSNP
rs943959385	547	dbSNP
rs989335274	548	dbSNP
rs1421813299	550	dbSNP
rs915157633	552	dbSNP
rs931945482	555	dbSNP
rs1474848490	564	dbSNP
rs144928076	566	dbSNP
rs911262700	567	dbSNP
rs944096630	580	dbSNP
rs1418873378	585	dbSNP
rs146756530	589	dbSNP
rs117682871	590	dbSNP
rs1013413346	592	dbSNP
rs540778125	602	dbSNP
rs1157264195	610	dbSNP
rs1205896629	612	dbSNP
rs749336842	617	dbSNP
rs1208742927	620	dbSNP
rs888230331	622	dbSNP
rs1344657946	625	dbSNP
rs199844502	628	dbSNP
rs1349700592	631	dbSNP
rs1280053680	632	dbSNP
rs1048586240	640	dbSNP
rs888678919	641	dbSNP
rs1444375775	644	dbSNP
rs1431661285	662	dbSNP
rs757333430	665	dbSNP
rs1461015904	675	dbSNP
rs1395455782	687	dbSNP
rs1167428752	688	dbSNP
rs780011412	690	dbSNP
rs1471805165	691	dbSNP
rs1407796227	699	dbSNP
rs1178142246	701	dbSNP
rs1291128571	708	dbSNP
rs1034457823	711	dbSNP
rs1245510023	711	dbSNP
rs895503211	719	dbSNP
rs1206498372	722	dbSNP
rs1316115596	724	dbSNP
rs1042426	731	dbSNP
rs970429801	733	dbSNP
rs1414709842	735	dbSNP
rs544532685	744	dbSNP
rs1030728349	745	dbSNP
rs879514353	759	dbSNP
rs1357783149	765	dbSNP
rs140372418	771	dbSNP
rs1328163149	773	dbSNP
rs1283165937	778	dbSNP
rs796365501	785	dbSNP
rs1217240978	786	dbSNP
rs1160231703	793	dbSNP
rs1402842416	797	dbSNP
rs1032420195	801	dbSNP
rs1022151039	805	dbSNP
rs953413303	809	dbSNP
rs1188598465	814	dbSNP
rs1254409106	815	dbSNP
rs530301650	816	dbSNP
rs986096709	820	dbSNP
rs911178884	825	dbSNP
rs1242369512	829	dbSNP
rs911190135	838	dbSNP
rs145258534	841	dbSNP
rs1279501341	846	dbSNP
rs548917050	853	dbSNP
rs776354186	861	dbSNP
rs1349966037	863	dbSNP
rs1175699745	864	dbSNP
rs1407609349	867	dbSNP
rs918508427	871	dbSNP
rs929790507	880	dbSNP
rs1401515432	889	dbSNP
rs75516158	890	dbSNP
rs1173559754	892	dbSNP
rs1435814631	895	dbSNP
rs368775209	896	dbSNP
rs567056452	897	dbSNP
rs866627781	900	dbSNP
rs1055563692	902	dbSNP
rs1314075048	906	dbSNP
rs1482148558	908	dbSNP
rs1256457131	910	dbSNP
rs1357721715	911	dbSNP
rs1049037567	919	dbSNP
rs1440545390	924	dbSNP
rs1272433508	928	dbSNP
rs895453538	938	dbSNP
rs1279353916	939	dbSNP
rs888655723	941	dbSNP
rs1225148564	942	dbSNP
rs948270379	946	dbSNP
rs747967905	952	dbSNP
rs1025179358	960	dbSNP
rs902808312	963	dbSNP
rs1306770900	971	dbSNP
rs1461032299	980	dbSNP
rs999636005	982	dbSNP
rs1211472856	987	dbSNP
rs564083044	989	dbSNP
rs1032947624	991	dbSNP
rs1045326131	995	dbSNP
rs1188344646	996	dbSNP
rs958248968	1003	dbSNP
rs1249503355	1004	dbSNP
rs1195219315	1018	dbSNP
rs1480349255	1022	dbSNP
rs906187700	1037	dbSNP
rs985919327	1038	dbSNP
rs1282474137	1039	dbSNP
rs1435855306	1040	dbSNP
rs1203482404	1041	dbSNP
rs1234254097	1043	dbSNP
rs1003177291	1049	dbSNP
rs1018368178	1050	dbSNP
rs1470609990	1054	dbSNP
rs1030654708	1056	dbSNP
rs1180764541	1058	dbSNP
rs528184500	1061	dbSNP
rs1393323617	1062	dbSNP
rs892196244	1063	dbSNP
rs1165259571	1064	dbSNP
rs1393239246	1064	dbSNP
rs1462807778	1065	dbSNP
rs1003459453	1066	dbSNP
rs965332536	1066	dbSNP
rs918497125	1067	dbSNP
rs1252607438	1069	dbSNP
rs546740776	1071	dbSNP
rs1272764446	1075	dbSNP
rs747382081	1080	dbSNP
rs1408719625	1084	dbSNP
rs571377603	1087	dbSNP
rs532430047	1088	dbSNP
rs1259270876	1091	dbSNP
rs1238805770	1093	dbSNP
rs1336826716	1097	dbSNP
rs1355504632	1097	dbSNP
rs1448506108	1101	dbSNP
rs1369685470	1105	dbSNP
rs909679569	1110	dbSNP
rs1369613136	1113	dbSNP
rs1442572079	1115	dbSNP
rs1382345928	1121	dbSNP
rs937198769	1123	dbSNP
rs1055914495	1125	dbSNP
rs1234461185	1129	dbSNP
rs8011	1130	dbSNP
rs1173932155	1134	dbSNP
rs1452650808	1135	dbSNP
rs147973565	1136	dbSNP
rs1207930840	1140	dbSNP
rs866002632	1146	dbSNP
rs1018881190	1147	dbSNP
rs1204884016	1154	dbSNP
rs14815	1162	dbSNP
rs1273383404	1169	dbSNP
rs1439394320	1171	dbSNP
rs965341967	1176	dbSNP
rs1200725879	1182	dbSNP
rs1263006511	1183	dbSNP
rs1251438640	1184	dbSNP
rs1347800802	1191	dbSNP
rs976731884	1199	dbSNP
rs1468078748	1201	dbSNP
rs1235886431	1205	dbSNP
rs1324281684	1212	dbSNP
rs181222302	1214	dbSNP
rs1379479279	1224	dbSNP
rs1386947938	1226	dbSNP
rs1000161772	1235	dbSNP
rs554673724	1241	dbSNP
rs1474685804	1243	dbSNP
rs930037457	1250	dbSNP
rs1167897306	1252	dbSNP
rs984230322	1261	dbSNP
rs527935415	1265	dbSNP
rs1479069756	1269	dbSNP
rs1420635248	1271	dbSNP
rs1467578641	1274	dbSNP
rs1447935782	1275	dbSNP
rs375778006	1277	dbSNP
rs948267699	1280	dbSNP
rs1045697448	1282	dbSNP
rs906782612	1287	dbSNP
rs1441182976	1288	dbSNP
rs111958889	1290	dbSNP
rs113187538	1291	dbSNP
rs939627032	1292	dbSNP
rs1336946207	1293	dbSNP

- Cell, 2010

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

- Nature methods, 2011

Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.

- Genes & development, 2011

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-beta signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area.

- Nature, 2013

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

- mBio, 2013

UNLABELLED: The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4(+) peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE: MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.