ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acAUCCGACCCCUAUUUACUUAu 5' || || | |||||||| Target 5' tgTATCCTTTTAAGAAATGAATa 3'	482 - 504	149.00	-7.90
2	miRNA 3' acaUCCGACCCCUAUUUACUUau 5' ||| | | | ||:|||| Target 5' atgAGGAGGAGTAAAAGTGAAat 3'	89 - 111	124.00	-8.50
3	miRNA 3' acauccGACCCCUA-------UUUACUUau 5' ||||| || ||||||: Target 5' attgttCTGGGTATTAGAAGAAAATGAGac 3'	427 - 456	121.00	-9.91

Mutant ID	Mutant Position	Mutant Source
COSN30503406	14	COSMIC
COSN510849	14	COSMIC
COSN30113546	26	COSMIC
COSN30101639	84	COSMIC
COSN30536154	116	COSMIC
COSN5763263	119	COSMIC
COSN28689105	155	COSMIC
COSN31588980	170	COSMIC
COSN32058129	243	COSMIC
COSN21439579	462	COSMIC
COSN22072404	480	COSMIC
COSN22065210	510	COSMIC
COSN32063556	602	COSMIC
COSN192087	1331	COSMIC
COSN21372555	1423	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs755255998	3	dbSNP
rs1804873	4	dbSNP
rs148357272	8	dbSNP
rs1048439798	9	dbSNP
rs912092660	14	dbSNP
rs557143425	15	dbSNP
rs376722538	16	dbSNP
rs992218658	17	dbSNP
rs780538803	18	dbSNP
rs747571736	20	dbSNP
rs1252381446	23	dbSNP
rs1160990657	29	dbSNP
rs780253134	30	dbSNP
rs771320672	32	dbSNP
rs769167200	34	dbSNP
rs781644374	34	dbSNP
rs746170581	37	dbSNP
rs1253820686	38	dbSNP
rs1435148182	40	dbSNP
rs1173968238	42	dbSNP
rs769501247	43	dbSNP
rs1343166018	44	dbSNP
rs1317542998	45	dbSNP
rs369190647	51	dbSNP
rs1161350290	52	dbSNP
rs1042768102	68	dbSNP
rs1327654333	69	dbSNP
rs774939168	80	dbSNP
rs140481516	87	dbSNP
rs1423852280	91	dbSNP
rs1383168063	95	dbSNP
rs1031986443	98	dbSNP
rs1445534088	103	dbSNP
rs1363407366	111	dbSNP
rs981094700	122	dbSNP
rs1439654576	124	dbSNP
rs543208640	136	dbSNP
rs959919430	167	dbSNP
rs745748608	170	dbSNP
rs1161577511	171	dbSNP
rs1489999755	180	dbSNP
rs1294491865	186	dbSNP
rs1392672419	191	dbSNP
rs562010934	192	dbSNP
rs1405736258	198	dbSNP
rs771592239	201	dbSNP
rs1236387063	204	dbSNP
rs1337025090	207	dbSNP
rs971685811	210	dbSNP
rs1343016532	215	dbSNP
rs1436653737	216	dbSNP
rs970134266	219	dbSNP
rs879883046	223	dbSNP
rs1297639953	239	dbSNP
rs1425039255	240	dbSNP
rs920643018	242	dbSNP
rs1051179	243	dbSNP
rs1244482260	246	dbSNP
rs933028533	248	dbSNP
rs150432263	250	dbSNP
rs1479766170	257	dbSNP
rs1271660057	262	dbSNP
rs1223820354	264	dbSNP
rs912165039	266	dbSNP
rs559370466	279	dbSNP
rs1282517386	284	dbSNP
rs1357572161	290	dbSNP
rs532763326	293	dbSNP
rs1282435458	297	dbSNP
rs912703936	306	dbSNP
rs942921172	309	dbSNP
rs932452768	316	dbSNP
rs1343616645	318	dbSNP
rs1040351700	323	dbSNP
rs1285139756	330	dbSNP
rs1043185227	341	dbSNP
rs182303853	342	dbSNP
rs904077784	351	dbSNP
rs1448886812	356	dbSNP
rs1315947247	359	dbSNP
rs1209469103	364	dbSNP
rs934336811	366	dbSNP
rs1242666747	367	dbSNP
rs1175857970	368	dbSNP
rs1001073869	376	dbSNP
rs1052756343	377	dbSNP
rs1460016052	382	dbSNP
rs1475342728	383	dbSNP
rs1240775700	385	dbSNP
rs569609468	386	dbSNP
rs1014142857	405	dbSNP
rs1268341570	405	dbSNP
rs1181750206	406	dbSNP
rs1052360413	408	dbSNP
rs1320841681	410	dbSNP
rs905689580	431	dbSNP
rs1219802184	435	dbSNP
rs1338663878	451	dbSNP
rs892498402	466	dbSNP
rs1013607217	472	dbSNP
rs994959261	478	dbSNP
rs1365603716	480	dbSNP
rs760414877	488	dbSNP
rs969621343	490	dbSNP
rs950807974	491	dbSNP
rs866623589	494	dbSNP
rs1409960441	500	dbSNP
rs1405380698	501	dbSNP
rs1417952402	505	dbSNP
rs111753262	511	dbSNP
rs1475958269	516	dbSNP
rs1320784078	527	dbSNP
rs963269719	534	dbSNP
rs971988626	536	dbSNP
rs1459655889	537	dbSNP
rs575550054	542	dbSNP
rs1449824085	551	dbSNP
rs1336779163	564	dbSNP
rs567655781	566	dbSNP
rs1306898920	567	dbSNP
rs1270825183	568	dbSNP
rs1216219182	570	dbSNP
rs954383431	574	dbSNP
rs1238781648	582	dbSNP
rs1278102302	585	dbSNP
rs987624501	589	dbSNP
rs1244136444	590	dbSNP
rs772185721	590	dbSNP
rs922132652	591	dbSNP
rs910335924	594	dbSNP
rs138245954	600	dbSNP
rs943118211	601	dbSNP
rs553225590	604	dbSNP
rs925705908	605	dbSNP
rs571326835	611	dbSNP
rs934391695	614	dbSNP
rs1052775318	638	dbSNP
rs193001907	643	dbSNP
rs1395556711	644	dbSNP
rs557433833	646	dbSNP
rs1460082732	652	dbSNP
rs1205577339	655	dbSNP
rs1162053926	657	dbSNP
rs1442439706	663	dbSNP
rs1039832491	669	dbSNP
rs1222557374	690	dbSNP
rs925471715	693	dbSNP
rs949984359	707	dbSNP
rs1044326362	709	dbSNP
rs1206559999	713	dbSNP
rs777108617	714	dbSNP
rs1219932346	719	dbSNP
rs1231010691	721	dbSNP
rs34341742	725	dbSNP
rs905718991	725	dbSNP
rs1052661394	726	dbSNP
rs1223391518	732	dbSNP
rs575647565	734	dbSNP
rs1371279968	743	dbSNP
rs1281296941	746	dbSNP
rs1411036047	752	dbSNP
rs1365474471	753	dbSNP
rs1473432039	756	dbSNP
rs1301736579	758	dbSNP
rs867742732	787	dbSNP
rs1350794271	790	dbSNP
rs1027462290	791	dbSNP
rs886531818	796	dbSNP
rs757942843	802	dbSNP
rs543644588	803	dbSNP
rs762261762	805	dbSNP
rs1046842380	806	dbSNP
rs1472898494	807	dbSNP
rs1018934490	808	dbSNP
rs966003995	828	dbSNP
rs974989443	828	dbSNP
rs1265741040	829	dbSNP
rs1215345185	836	dbSNP
rs1315336335	840	dbSNP
rs1428144279	858	dbSNP
rs905238354	861	dbSNP
rs968298424	862	dbSNP
rs1229060126	865	dbSNP
rs1002717887	868	dbSNP
rs544540889	878	dbSNP
rs1281965339	893	dbSNP
rs1403576790	895	dbSNP
rs765627147	896	dbSNP
rs1288646309	904	dbSNP
rs963237124	910	dbSNP
rs993394954	911	dbSNP
rs1343037603	915	dbSNP
rs1160802208	916	dbSNP
rs1391375260	918	dbSNP
rs1327406656	922	dbSNP
rs1427936719	925	dbSNP
rs750976916	927	dbSNP
rs934446307	930	dbSNP
rs1410814577	933	dbSNP
rs1185708663	934	dbSNP
rs1260787038	935	dbSNP
rs1209552950	937	dbSNP
rs988552632	938	dbSNP
rs143829329	941	dbSNP
rs1321594593	948	dbSNP
rs1235553377	950	dbSNP
rs1256611779	951	dbSNP
rs1338799350	952	dbSNP
rs1195197843	954	dbSNP
rs1272383343	955	dbSNP
rs1313296581	955	dbSNP
rs571795401	956	dbSNP
rs1364460121	957	dbSNP
rs1182555485	966	dbSNP
rs183383393	970	dbSNP
rs927218114	972	dbSNP
rs910210602	975	dbSNP
rs964467357	979	dbSNP
rs990924111	982	dbSNP
rs978929475	983	dbSNP
rs925440510	989	dbSNP
rs1378101781	994	dbSNP
rs1169597553	1002	dbSNP
rs1426554256	1006	dbSNP
rs1189473503	1009	dbSNP
rs1249594600	1009	dbSNP
rs1479227669	1011	dbSNP
rs1049405188	1015	dbSNP
rs1241439812	1017	dbSNP
rs886264286	1033	dbSNP
rs936882398	1037	dbSNP
rs1483890603	1038	dbSNP
rs1225819255	1043	dbSNP
rs1309635385	1045	dbSNP
rs1298718134	1050	dbSNP
rs1377741313	1055	dbSNP
rs1381577830	1058	dbSNP
rs1334167861	1059	dbSNP
rs11483279	1065	dbSNP
rs397866099	1065	dbSNP
rs1040415791	1067	dbSNP
rs1166725469	1068	dbSNP
rs541358891	1072	dbSNP
rs1168988832	1073	dbSNP
rs1444283721	1080	dbSNP
rs1383598800	1086	dbSNP
rs1189815126	1088	dbSNP
rs1424501766	1090	dbSNP
rs1430810229	1092	dbSNP
rs1194596603	1099	dbSNP
rs1302884796	1100	dbSNP
rs901912078	1104	dbSNP
rs1388793148	1106	dbSNP
rs1222173009	1107	dbSNP
rs559652212	1113	dbSNP
rs1028915639	1114	dbSNP
rs1246449422	1114	dbSNP
rs532950343	1115	dbSNP
rs968707108	1123	dbSNP
rs1389672983	1127	dbSNP
rs2423999	1127	dbSNP
rs1372299320	1130	dbSNP
rs1308466000	1132	dbSNP
rs1302207085	1135	dbSNP
rs560393065	1135	dbSNP
rs1325999024	1137	dbSNP
rs1363354125	1142	dbSNP
rs1179742966	1145	dbSNP
rs914123990	1150	dbSNP
rs543410065	1160	dbSNP
rs532592278	1161	dbSNP
rs1199278590	1178	dbSNP
rs147255550	1184	dbSNP
rs988583658	1185	dbSNP
rs77456063	1188	dbSNP
rs971109609	1195	dbSNP
rs1259676241	1196	dbSNP
rs979774584	1199	dbSNP
rs1351751819	1202	dbSNP
rs1288939932	1208	dbSNP
rs549197561	1211	dbSNP
rs1205645600	1232	dbSNP
rs926940932	1239	dbSNP
rs73900626	1242	dbSNP
rs1325261766	1243	dbSNP
rs145960850	1248	dbSNP
rs1211338947	1252	dbSNP
rs778316010	1253	dbSNP
rs946715786	1254	dbSNP
rs1267600921	1256	dbSNP
rs1406239211	1265	dbSNP
rs1037812983	1266	dbSNP
rs1384486882	1272	dbSNP
rs3171297	1276	dbSNP
rs993365169	1287	dbSNP
rs1186048773	1288	dbSNP
rs771474550	1292	dbSNP
rs758000104	1293	dbSNP
rs1009245656	1295	dbSNP
rs142942865	1296	dbSNP
rs546902587	1299	dbSNP
rs1248888449	1300	dbSNP
rs1163479160	1301	dbSNP
rs1199992609	1321	dbSNP
rs1017240689	1324	dbSNP
rs1364140817	1330	dbSNP
rs1451876579	1334	dbSNP
rs1260640919	1341	dbSNP
rs964759814	1343	dbSNP
rs978533880	1346	dbSNP
rs1268139876	1348	dbSNP
rs1032759009	1350	dbSNP
rs879698128	1356	dbSNP
rs1276437972	1357	dbSNP
rs1384450618	1359	dbSNP
rs373777333	1369	dbSNP
rs1380605564	1374	dbSNP
rs35465475	1382	dbSNP
rs988232268	1383	dbSNP
rs901943133	1403	dbSNP
rs571564065	1406	dbSNP
rs1389548078	1408	dbSNP
rs1336160201	1412	dbSNP
rs1468741227	1415	dbSNP
rs1367291445	1417	dbSNP
rs377509976	1418	dbSNP
rs138910697	1419	dbSNP
rs926626840	1420	dbSNP
rs1202452402	1423	dbSNP
rs1031235113	1428	dbSNP
rs938049535	1429	dbSNP
rs1037780962	1432	dbSNP
rs1219696866	1435	dbSNP
rs1025115922	1436	dbSNP
rs550691745	1438	dbSNP
rs1230973963	1439	dbSNP
rs929378172	1441	dbSNP
rs188692522	1442	dbSNP
rs1336215459	1448	dbSNP
rs890296820	1450	dbSNP
rs1206310731	1451	dbSNP
rs926978886	1456	dbSNP
rs1249404438	1457	dbSNP
rs1464929525	1470	dbSNP
rs1360921310	1471	dbSNP
rs951725783	1473	dbSNP
rs1409842427	1474	dbSNP
rs1412241026	1483	dbSNP
rs1008801311	1488	dbSNP
rs536327215	1490	dbSNP
rs900159696	1493	dbSNP
rs555073191	1495	dbSNP
rs1390381076	1501	dbSNP
rs1165846999	1502	dbSNP
rs907797833	1509	dbSNP
rs369627547	1516	dbSNP
rs1210803736	1517	dbSNP
rs757058559	1518	dbSNP
rs1322779551	1525	dbSNP
rs1032644827	1526	dbSNP
rs955766571	1528	dbSNP
rs988534086	1529	dbSNP
rs371383650	1536	dbSNP
rs971328648	1544	dbSNP
rs1314665930	1556	dbSNP
rs1336015731	1558	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.

- 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.

- Genome biology, 2014

BACKGROUND: Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference. RESULTS: To generate a biochemically-validated set of miRNA-binding sites, we performed argonaute-2 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO2-PAR-CLIP) in MCF7 cells. We then defined putative miRNA-target interactions using a computational model, which ranked and selected additional TargetScan-predicted interactions based on features of our AGO2-PAR-CLIP binding-site data. We subselected modeled interactions according to the abundance of their constituent miRNA and mRNA transcripts in tumors, and we took advantage of the variability of miRNA expression within molecular subtypes to detect miRNA repression. Interestingly, our data suggest that miRNA families control subtype-specific pathways; for example, miR-17, miR-19a, miR-25, and miR-200b show high miRNA regulatory activity in the triple-negative, basal-like subtype, whereas miR-22 and miR-24 do so in the HER2 subtype. An independent dataset validated our findings for miR-17 and miR-25, and showed a correlation between the expression levels of miR-182 targets and overall patient survival. Pathway analysis associated miR-17, miR-19a, and miR-200b with leukocyte transendothelial migration. CONCLUSIONS: We combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.