ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agUGUA-AACGGACGUCUCUAAa 5' | || ||||| ||||||| Target 5' cgAAATATTGCC--AAGAGATTg 3'	625 - 645	156.00	-12.10
2	miRNA 3' agUGUAAACGGACGUCUCUAAa 5' |: ||| :|| ||||||| Target 5' tcATCTTTATCT--AGAGATTg 3'	796 - 815	156.00	-9.50
3	miRNA 3' agUGUAAACGGACG-UCUCUaaa 5' ||| |||: ||: ||||| Target 5' caACA-TTGTATGTGAGAGAaat 3'	1009 - 1030	123.00	-9.02

Mutant ID	Mutant Position	Mutant Source
COSN30694458	54	COSMIC
COSN19644387	87	COSMIC
COSN28080334	143	COSMIC
COSN1340339	154	COSMIC
COSN30172585	174	COSMIC
COSN25001297	208	COSMIC
COSN28880340	225	COSMIC
COSN1340341	260	COSMIC
COSN26093681	278	COSMIC
COSN31568450	297	COSMIC
COSN31526410	388	COSMIC
COSN29163215	406	COSMIC
COSN29859024	537	COSMIC
COSN31520161	690	COSMIC
COSN9918899	696	COSMIC
COSN6343020	741	COSMIC
COSN31553404	759	COSMIC
COSN31551432	832	COSMIC
COSN17875949	933	COSMIC
COSN15957897	1005	COSMIC
COSN7919908	1106	COSMIC
COSN25463944	1145	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs770071519	8	dbSNP
rs539029657	10	dbSNP
rs763361103	12	dbSNP
rs752039544	13	dbSNP
rs187252724	15	dbSNP
rs760165628	18	dbSNP
rs765822549	19	dbSNP
rs753446787	20	dbSNP
rs754671964	21	dbSNP
rs778748143	25	dbSNP
rs752517500	27	dbSNP
rs1261691651	28	dbSNP
rs371758437	29	dbSNP
rs945316053	32	dbSNP
rs1489346271	33	dbSNP
rs375296782	37	dbSNP
rs1403805932	38	dbSNP
rs961770465	40	dbSNP
rs1280745078	42	dbSNP
rs1254002403	45	dbSNP
rs368994075	45	dbSNP
rs770982059	46	dbSNP
rs781130127	49	dbSNP
rs1349056499	50	dbSNP
rs1294839027	51	dbSNP
rs934325936	53	dbSNP
rs1341765526	60	dbSNP
rs1469157139	61	dbSNP
rs1298315130	77	dbSNP
rs1156265127	80	dbSNP
rs1304933870	81	dbSNP
rs1466939369	92	dbSNP
rs1372997382	94	dbSNP
rs1168883960	103	dbSNP
rs192021096	110	dbSNP
rs1424360343	123	dbSNP
rs1054098392	125	dbSNP
rs1173569279	128	dbSNP
rs3763457	130	dbSNP
rs748551409	134	dbSNP
rs1412129891	139	dbSNP
rs996964896	139	dbSNP
rs763978989	140	dbSNP
rs1049615597	141	dbSNP
rs11408141	142	dbSNP
rs1232197871	142	dbSNP
rs1396015761	142	dbSNP
rs771340615	142	dbSNP
rs890863270	143	dbSNP
rs1396265365	146	dbSNP
rs1009053743	150	dbSNP
rs1335437829	154	dbSNP
rs1440327394	155	dbSNP
rs1276638611	156	dbSNP
rs1008168399	165	dbSNP
rs1345819313	166	dbSNP
rs1234930381	173	dbSNP
rs1279597781	178	dbSNP
rs1019066720	190	dbSNP
rs1164298542	191	dbSNP
rs962479862	198	dbSNP
rs1474300138	207	dbSNP
rs1370282439	211	dbSNP
rs1188424361	221	dbSNP
rs993770405	224	dbSNP
rs1028291555	226	dbSNP
rs751476171	227	dbSNP
rs757027606	228	dbSNP
rs980756568	229	dbSNP
rs926731037	230	dbSNP
rs1459010612	238	dbSNP
rs1258710202	242	dbSNP
rs1436816977	250	dbSNP
rs868648101	255	dbSNP
rs1179240963	257	dbSNP
rs1353578590	259	dbSNP
rs960781352	260	dbSNP
rs995470876	261	dbSNP
rs1334850837	271	dbSNP
rs1193543926	274	dbSNP
rs1026909685	289	dbSNP
rs1431981149	294	dbSNP
rs73428112	304	dbSNP
rs377765156	306	dbSNP
rs1325750961	314	dbSNP
rs1392922910	317	dbSNP
rs1408364477	317	dbSNP
rs3763456	323	dbSNP
rs945176171	327	dbSNP
rs1453295538	328	dbSNP
rs3763455	335	dbSNP
rs147964164	356	dbSNP
rs1302554627	362	dbSNP
rs978256942	373	dbSNP
rs773461640	380	dbSNP
rs1368653320	388	dbSNP
rs932363014	389	dbSNP
rs577345709	399	dbSNP
rs890914260	401	dbSNP
rs1209952260	403	dbSNP
rs1007933017	408	dbSNP
rs1241889425	412	dbSNP
rs546198697	420	dbSNP
rs1338513118	425	dbSNP
rs546419128	426	dbSNP
rs1434882573	433	dbSNP
rs1333383091	436	dbSNP
rs1324250528	439	dbSNP
rs1193715570	441	dbSNP
rs1054127719	446	dbSNP
rs1358850207	446	dbSNP
rs1291649357	450	dbSNP
rs183438831	452	dbSNP
rs914230809	459	dbSNP
rs1476980861	460	dbSNP
rs932457785	465	dbSNP
rs1212566956	469	dbSNP
rs1198932984	470	dbSNP
rs1445859992	471	dbSNP
rs1260414009	481	dbSNP
rs188843857	487	dbSNP
rs1218287015	493	dbSNP
rs542069377	494	dbSNP
rs1036703400	504	dbSNP
rs562265068	505	dbSNP
rs531133352	508	dbSNP
rs1194529330	509	dbSNP
rs954326608	515	dbSNP
rs890989263	522	dbSNP
rs994047435	527	dbSNP
rs1027937532	528	dbSNP
rs888020129	533	dbSNP
rs1036786604	538	dbSNP
rs897087497	539	dbSNP
rs1375108939	541	dbSNP
rs1002247238	542	dbSNP
rs1033685052	551	dbSNP
rs1412598149	552	dbSNP
rs1395549244	554	dbSNP
rs550830726	557	dbSNP
rs1173022178	559	dbSNP
rs1026983375	561	dbSNP
rs1461154159	564	dbSNP
rs1323918384	569	dbSNP
rs566651601	572	dbSNP
rs895121661	573	dbSNP
rs992258453	584	dbSNP
rs1390942725	590	dbSNP
rs1437107554	595	dbSNP
rs1444434522	603	dbSNP
rs1021435242	607	dbSNP
rs1195693004	609	dbSNP
rs1316623144	626	dbSNP
rs1296296589	627	dbSNP
rs1280135495	628	dbSNP
rs1012362688	632	dbSNP
rs1355040172	638	dbSNP
rs1282580865	639	dbSNP
rs570785153	640	dbSNP
rs1450469367	647	dbSNP
rs4727712	649	dbSNP
rs1225028602	650	dbSNP
rs1285559795	656	dbSNP
rs1334592688	664	dbSNP
rs1335031411	666	dbSNP
rs1025495174	683	dbSNP
rs779654673	694	dbSNP
rs748832814	700	dbSNP
rs559356585	706	dbSNP
rs1412466511	710	dbSNP
rs1179687514	716	dbSNP
rs768170424	717	dbSNP
rs1473594610	719	dbSNP
rs538965165	723	dbSNP
rs778162838	737	dbSNP
rs760865152	741	dbSNP
rs978666068	744	dbSNP
rs1178451536	747	dbSNP
rs1456819158	748	dbSNP
rs1260606572	754	dbSNP
rs1199832642	756	dbSNP
rs1031138301	762	dbSNP
rs958308561	765	dbSNP
rs747491232	767	dbSNP
rs1230189341	770	dbSNP
rs932566447	771	dbSNP
rs1284540781	775	dbSNP
rs1445861892	778	dbSNP
rs1374555859	779	dbSNP
rs985302952	784	dbSNP
rs912235150	789	dbSNP
rs914263682	789	dbSNP
rs943676756	794	dbSNP
rs879022721	800	dbSNP
rs1036799177	809	dbSNP
rs1445609783	814	dbSNP
rs879341784	814	dbSNP
rs1158402367	819	dbSNP
rs985113806	819	dbSNP
rs1438152451	820	dbSNP
rs912303300	823	dbSNP
rs1179768550	824	dbSNP
rs1488452983	827	dbSNP
rs1245030455	828	dbSNP
rs1322551752	830	dbSNP
rs1190006829	833	dbSNP
rs1419064494	838	dbSNP
rs1267359009	842	dbSNP
rs1474970388	843	dbSNP
rs896886017	843	dbSNP
rs1036859926	849	dbSNP
rs896991102	853	dbSNP
rs1366888053	859	dbSNP
rs1291280788	860	dbSNP
rs929526223	863	dbSNP
rs1163058188	864	dbSNP
rs931255458	873	dbSNP
rs1382503156	874	dbSNP
rs1159677479	881	dbSNP
rs1433079515	885	dbSNP
rs193080310	887	dbSNP
rs1423767353	902	dbSNP
rs1049694242	903	dbSNP
rs1454426769	910	dbSNP
rs1488815630	911	dbSNP
rs546176562	916	dbSNP
rs566451900	917	dbSNP
rs1380489147	919	dbSNP
rs1287868615	925	dbSNP
rs1335883755	927	dbSNP
rs879197346	928	dbSNP
rs1024934093	944	dbSNP
rs1033902627	946	dbSNP
rs1220873116	950	dbSNP
rs896507951	953	dbSNP
rs141839050	957	dbSNP
rs1397724320	961	dbSNP
rs1362108756	962	dbSNP
rs1021066578	963	dbSNP
rs1031211821	965	dbSNP
rs958173825	980	dbSNP
rs1239190141	987	dbSNP
rs989759587	993	dbSNP
rs1434287507	994	dbSNP
rs1422953650	997	dbSNP
rs1418030987	1004	dbSNP
rs1259667829	1008	dbSNP
rs1478909264	1011	dbSNP
rs1349107108	1021	dbSNP
rs753053113	1033	dbSNP
rs555294355	1036	dbSNP
rs1254056880	1046	dbSNP
rs1258576676	1047	dbSNP
rs1212089295	1050	dbSNP
rs1223060193	1055	dbSNP
rs147039924	1055	dbSNP
rs538024556	1056	dbSNP
rs1216327357	1066	dbSNP
rs985539760	1068	dbSNP
rs953844769	1069	dbSNP
rs777202119	1077	dbSNP
rs1485589098	1080	dbSNP
rs1377381430	1092	dbSNP
rs943824186	1098	dbSNP
rs985355283	1107	dbSNP
rs558113219	1109	dbSNP
rs1400270025	1110	dbSNP
rs912454372	1111	dbSNP
rs943728970	1118	dbSNP
rs1168623045	1125	dbSNP
rs756720560	1127	dbSNP
rs1186061100	1130	dbSNP
rs1467194651	1132	dbSNP
rs138263970	1134	dbSNP
rs1187052377	1135	dbSNP
rs2286259	1136	dbSNP
rs1048193710	1141	dbSNP
rs1183418956	1149	dbSNP
rs1461709567	1158	dbSNP
rs895056079	1159	dbSNP
rs1196688904	1162	dbSNP
rs947960741	1170	dbSNP
rs1251417595	1172	dbSNP
rs931061423	1185	dbSNP
rs1319607990	1186	dbSNP
rs1175438856	1189	dbSNP
rs1048112028	1190	dbSNP
rs1453921787	1197	dbSNP
rs1376640954	1207	dbSNP
rs887958331	1213	dbSNP
rs553294284	1224	dbSNP
rs1375831917	1225	dbSNP
rs906530414	1231	dbSNP
rs938138708	1242	dbSNP
rs999634007	1244	dbSNP
rs1052505746	1248	dbSNP
rs1055409709	1249	dbSNP
rs1166632075	1256	dbSNP
rs575130641	1264	dbSNP
rs1375019336	1266	dbSNP
rs1013625858	1269	dbSNP
rs1011097455	1270	dbSNP
rs1176660478	1275	dbSNP
rs1042779390	1281	dbSNP
rs1250592651	1282	dbSNP
rs754279101	1286	dbSNP
rs1391641431	1289	dbSNP
rs35515962	1294	dbSNP
rs113202530	1303	dbSNP
rs1266904276	1314	dbSNP
rs1001363431	1317	dbSNP
rs1333483350	1323	dbSNP
rs1236065506	1327	dbSNP
rs1353383632	1329	dbSNP
rs542283661	1333	dbSNP
rs562161357	1334	dbSNP
rs1336910012	1337	dbSNP
rs953897218	1341	dbSNP
rs1006658736	1351	dbSNP
rs1385219696	1356	dbSNP
rs185819843	1369	dbSNP
rs972345109	1380	dbSNP
rs1419362912	1381	dbSNP
rs965265264	1382	dbSNP
rs1156668322	1397	dbSNP
rs1441437154	1400	dbSNP
rs1176851722	1406	dbSNP
rs1248421862	1408	dbSNP
rs865909211	1412	dbSNP
rs757399391	1418	dbSNP
rs1197538904	1428	dbSNP
rs918356755	1431	dbSNP
rs80193567	1434	dbSNP
rs1195410222	1442	dbSNP
rs952384511	1444	dbSNP
rs143312141	1445	dbSNP
rs1434064656	1449	dbSNP
rs1219331940	1452	dbSNP
rs927011748	1453	dbSNP
rs916406981	1461	dbSNP
rs1046291804	1470	dbSNP
rs948002849	1470	dbSNP
rs533402952	1473	dbSNP
rs1356079691	1486	dbSNP
rs762977057	1487	dbSNP
rs1297319356	1488	dbSNP
rs935331177	1489	dbSNP
rs1052411161	1492	dbSNP
rs1393679275	1494	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.

- 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.