ID	Duplex structure	Position	Score	MFE
1	miRNA 3' auUUGGAUGUCCGUCCUGAUu 5' |||:| | |||||||| Target 5' taAACTT---GACAGGACTAt 3'	196 - 213	156.00	-15.80
2	miRNA 3' auUUGGAUGUCCGUCCUGAUu 5' || ||:||||::||: || Target 5' aaAAGCTGCAGGTGGGGATAa 3'	261 - 281	115.00	-19.40
3	miRNA 3' auuUGG--AUGUC-CGUCCUGAuu 5' ||: || || ::||||| Target 5' cacACTTGTAAAGTTTGGGACTta 3'	8 - 31	112.00	-8.80

Mutant ID	Mutant Position	Mutant Source
COSN13375597	5	COSMIC
COSN8585505	5	COSMIC
COSN26993501	12	COSMIC
COSN1560442	24	COSMIC
COSN30193722	29	COSMIC
COSN30189211	35	COSMIC
COSN30130534	38	COSMIC
COSN30737548	101	COSMIC
COSN31549272	114	COSMIC
COSN28735540	146	COSMIC
COSN31525292	148	COSMIC
COSN1560440	212	COSMIC
COSN31486457	287	COSMIC
COSN5713346	352	COSMIC
COSN31484152	385	COSMIC
COSN17731134	636	COSMIC
COSN28735724	644	COSMIC
COSN28794452	662	COSMIC
COSN8585504	762	COSMIC
COSN28796952	839	COSMIC
COSN19373171	986	COSMIC
COSN5933049	1306	COSMIC
COSN22631148	2031	COSMIC
rs12818552	1231	GWAS

Mutant ID	Mutant Position	Mutant Source
rs754121347	1	dbSNP
rs1430082972	2	dbSNP
rs780370347	3	dbSNP
rs1314879694	4	dbSNP
rs542262304	5	dbSNP
rs1366166063	7	dbSNP
rs1228009284	12	dbSNP
rs1350053406	16	dbSNP
rs762206849	16	dbSNP
rs1171505089	20	dbSNP
rs750595710	21	dbSNP
rs1278519147	23	dbSNP
rs1420103757	30	dbSNP
rs1115154	32	dbSNP
rs1243101213	33	dbSNP
rs375470822	35	dbSNP
rs1187496822	37	dbSNP
rs1161110290	39	dbSNP
rs763303982	42	dbSNP
rs776423338	42	dbSNP
rs763831459	45	dbSNP
rs1428546294	46	dbSNP
rs555480464	49	dbSNP
rs762849688	49	dbSNP
rs1327693397	54	dbSNP
rs900292884	55	dbSNP
rs773607957	64	dbSNP
rs1053458365	65	dbSNP
rs1038744255	67	dbSNP
rs1373537281	69	dbSNP
rs1437274932	71	dbSNP
rs745957489	79	dbSNP
rs1343460916	81	dbSNP
rs942893969	102	dbSNP
rs1386167320	103	dbSNP
rs146283745	105	dbSNP
rs902174654	110	dbSNP
rs544754927	113	dbSNP
rs780296834	118	dbSNP
rs986957832	119	dbSNP
rs1178840855	120	dbSNP
rs1412636073	127	dbSNP
rs530691474	129	dbSNP
rs946298346	130	dbSNP
rs943958818	135	dbSNP
rs912400118	136	dbSNP
rs1168731128	142	dbSNP
rs913444483	142	dbSNP
rs990594092	143	dbSNP
rs1049528730	145	dbSNP
rs1323161255	146	dbSNP
rs571488556	149	dbSNP
rs141829241	151	dbSNP
rs537269086	156	dbSNP
rs1198722313	158	dbSNP
rs879391149	162	dbSNP
rs1226942685	173	dbSNP
rs558763629	173	dbSNP
rs940052738	187	dbSNP
rs1358002437	188	dbSNP
rs908519837	190	dbSNP
rs1210013721	194	dbSNP
rs982129605	201	dbSNP
rs1255255278	206	dbSNP
rs1197222468	211	dbSNP
rs1449310515	212	dbSNP
rs748605669	214	dbSNP
rs551302444	222	dbSNP
rs1252443051	223	dbSNP
rs1023156506	225	dbSNP
rs190177498	226	dbSNP
rs1034462574	227	dbSNP
rs957519306	237	dbSNP
rs1032134481	245	dbSNP
rs1473021048	252	dbSNP
rs1000599295	254	dbSNP
rs1388867014	258	dbSNP
rs14519	263	dbSNP
rs1411719231	271	dbSNP
rs1214798794	272	dbSNP
rs1308784824	275	dbSNP
rs1340806005	275	dbSNP
rs1370452183	278	dbSNP
rs902206238	281	dbSNP
rs1024160123	288	dbSNP
rs1020592007	289	dbSNP
rs111226568	292	dbSNP
rs891007370	302	dbSNP
rs1353899780	303	dbSNP
rs781737893	303	dbSNP
rs932518821	304	dbSNP
rs1482455234	309	dbSNP
rs1027572122	310	dbSNP
rs994938594	313	dbSNP
rs11548718	315	dbSNP
rs1442727396	322	dbSNP
rs554238720	323	dbSNP
rs1038650701	332	dbSNP
rs1444236584	334	dbSNP
rs1451604749	339	dbSNP
rs1169277220	340	dbSNP
rs1399281534	344	dbSNP
rs898329470	352	dbSNP
rs1329745594	356	dbSNP
rs1427943881	361	dbSNP
rs1411889961	367	dbSNP
rs1315279644	372	dbSNP
rs1038438422	381	dbSNP
rs940104788	386	dbSNP
rs1172109703	387	dbSNP
rs1332650832	388	dbSNP
rs1008612168	391	dbSNP
rs1455084811	394	dbSNP
rs908549547	395	dbSNP
rs868147931	397	dbSNP
rs1340758531	399	dbSNP
rs981442267	400	dbSNP
rs1177324531	402	dbSNP
rs1457541645	405	dbSNP
rs1345717129	406	dbSNP
rs950350751	408	dbSNP
rs1199635004	417	dbSNP
rs916200297	419	dbSNP
rs991739055	420	dbSNP
rs57347146	425	dbSNP
rs1466108183	426	dbSNP
rs1189414813	433	dbSNP
rs1247651213	437	dbSNP
rs1447675795	438	dbSNP
rs3741448	440	dbSNP
rs1033165665	441	dbSNP
rs1466019033	445	dbSNP
rs1275856050	449	dbSNP
rs1171354988	453	dbSNP
rs1391240362	454	dbSNP
rs1218199698	458	dbSNP
rs979605885	468	dbSNP
rs1348486964	476	dbSNP
rs28701727	483	dbSNP
rs1021044623	487	dbSNP
rs980071740	488	dbSNP
rs1232379875	491	dbSNP
rs1007862164	494	dbSNP
rs550251854	500	dbSNP
rs1330870230	509	dbSNP
rs1209140515	518	dbSNP
rs1279976637	519	dbSNP
rs890782683	520	dbSNP
rs1192967645	521	dbSNP
rs971481218	522	dbSNP
rs538390285	541	dbSNP
rs1028234852	542	dbSNP
rs1458081050	555	dbSNP
rs1158981808	556	dbSNP
rs1316694210	558	dbSNP
rs1357714622	559	dbSNP
rs1333581474	561	dbSNP
rs1294948811	565	dbSNP
rs996697965	571	dbSNP
rs570907041	572	dbSNP
rs1299034751	575	dbSNP
rs950563668	578	dbSNP
rs1287539740	580	dbSNP
rs1326839467	580	dbSNP
rs1377354374	586	dbSNP
rs1266267526	589	dbSNP
rs1157338366	594	dbSNP
rs552745240	601	dbSNP
rs527941859	603	dbSNP
rs1028005286	605	dbSNP
rs1439642806	606	dbSNP
rs1254585996	608	dbSNP
rs1409145923	609	dbSNP
rs1473229229	610	dbSNP
rs1416514969	614	dbSNP
rs1179359621	617	dbSNP
rs746046095	618	dbSNP
rs1235438652	619	dbSNP
rs778414079	621	dbSNP
rs185268566	622	dbSNP
rs1350126040	630	dbSNP
rs548929406	631	dbSNP
rs887159040	635	dbSNP
rs1046153729	636	dbSNP
rs950119458	639	dbSNP
rs1300820601	649	dbSNP
rs1008477700	650	dbSNP
rs10998	662	dbSNP
rs1056087950	667	dbSNP
rs528248833	671	dbSNP
rs1370820129	674	dbSNP
rs1010851152	682	dbSNP
rs1321419025	684	dbSNP
rs1284690756	686	dbSNP
rs868765017	690	dbSNP
rs1483436233	691	dbSNP
rs562912213	694	dbSNP
rs182225742	702	dbSNP
rs1250259134	706	dbSNP
rs1484411203	707	dbSNP
rs926130156	716	dbSNP
rs1414066620	718	dbSNP
rs1422926889	722	dbSNP
rs977579768	725	dbSNP
rs966871512	733	dbSNP
rs1391432998	742	dbSNP
rs1283747637	748	dbSNP
rs1307275159	751	dbSNP
rs1415655248	761	dbSNP
rs1352750836	763	dbSNP
rs1357389152	765	dbSNP
rs757298847	783	dbSNP
rs1303252029	792	dbSNP
rs1419592789	793	dbSNP
rs1450660832	795	dbSNP
rs1346376459	801	dbSNP
rs913687913	805	dbSNP
rs1300137166	811	dbSNP
rs1161717186	812	dbSNP
rs986950998	816	dbSNP
rs1227786639	817	dbSNP
rs1271492864	818	dbSNP
rs1314536906	823	dbSNP
rs1211227689	829	dbSNP
rs955063795	837	dbSNP
rs4758660	839	dbSNP
rs996730484	842	dbSNP
rs1249515768	850	dbSNP
rs777755888	854	dbSNP
rs1017181587	857	dbSNP
rs1417259579	873	dbSNP
rs1004015174	876	dbSNP
rs1416153436	877	dbSNP
rs950835845	878	dbSNP
rs886933983	879	dbSNP
rs1426617527	881	dbSNP
rs1322745252	888	dbSNP
rs1366276599	888	dbSNP
rs920451684	888	dbSNP
rs973175151	888	dbSNP
rs1045839389	891	dbSNP
rs1369843954	893	dbSNP
rs757619621	896	dbSNP
rs964600684	900	dbSNP
rs1321774921	907	dbSNP
rs1217864774	908	dbSNP
rs1014741998	912	dbSNP
rs145997829	913	dbSNP
rs1234103338	914	dbSNP
rs1187362857	915	dbSNP
rs1239814118	923	dbSNP
rs1478743888	927	dbSNP
rs1337274291	928	dbSNP
rs1424391992	931	dbSNP
rs1276378050	936	dbSNP
rs1468029344	937	dbSNP
rs547594751	942	dbSNP
rs1021146359	943	dbSNP
rs767505909	945	dbSNP
rs936243462	949	dbSNP
rs755001584	950	dbSNP
rs1009338378	951	dbSNP
rs1437293379	954	dbSNP
rs751730120	956	dbSNP
rs893519680	957	dbSNP
rs946273177	961	dbSNP
rs1033190667	962	dbSNP
rs1000327871	966	dbSNP
rs1328933009	968	dbSNP
rs766480987	971	dbSNP
rs906155928	975	dbSNP
rs1281703335	977	dbSNP
rs1452777794	977	dbSNP
rs912027086	988	dbSNP
rs1268095841	993	dbSNP
rs1479439933	998	dbSNP
rs540444061	1001	dbSNP
rs1412381519	1003	dbSNP
rs1472143636	1008	dbSNP
rs763211350	1013	dbSNP
rs1393214921	1018	dbSNP
rs1406021723	1022	dbSNP
rs371902028	1028	dbSNP
rs895832780	1029	dbSNP
rs920947607	1030	dbSNP
rs1048162182	1034	dbSNP
rs1487491524	1035	dbSNP
rs929413313	1036	dbSNP
rs527363263	1037	dbSNP
rs973249325	1040	dbSNP
rs1290467051	1041	dbSNP
rs1256332806	1044	dbSNP
rs1207125402	1047	dbSNP
rs1226971876	1048	dbSNP
rs1308103179	1056	dbSNP
rs962609449	1057	dbSNP
rs572979860	1063	dbSNP
rs1297878807	1069	dbSNP
rs1252466569	1070	dbSNP
rs1217948127	1075	dbSNP
rs1370999812	1078	dbSNP
rs1179475181	1079	dbSNP
rs1370384541	1082	dbSNP
rs765545173	1082	dbSNP
rs762168805	1083	dbSNP
rs910497632	1084	dbSNP
rs1443496507	1089	dbSNP
rs1004519178	1096	dbSNP
rs1431711556	1099	dbSNP
rs1169721655	1102	dbSNP
rs1398568871	1107	dbSNP
rs1328816398	1110	dbSNP
rs1321277868	1112	dbSNP
rs987331597	1113	dbSNP
rs776943815	1115	dbSNP
rs1341874799	1118	dbSNP
rs1448243778	1120	dbSNP
rs1373127510	1121	dbSNP
rs1356564287	1130	dbSNP
rs1230046460	1131	dbSNP
rs954764597	1134	dbSNP
rs1193835125	1138	dbSNP
rs112937401	1146	dbSNP
rs1260094210	1148	dbSNP
rs1014367124	1149	dbSNP
rs561805076	1150	dbSNP
rs574888547	1151	dbSNP
rs1194199364	1153	dbSNP
rs988179352	1155	dbSNP
rs957816546	1157	dbSNP
rs1372980058	1159	dbSNP
rs894526017	1160	dbSNP
rs1469594773	1161	dbSNP
rs1403311692	1162	dbSNP
rs1414268834	1162	dbSNP
rs745363367	1166	dbSNP
rs1380204699	1170	dbSNP
rs1436602621	1172	dbSNP
rs1032093519	1175	dbSNP
rs1195422470	1191	dbSNP
rs1257692921	1199	dbSNP
rs115378036	1200	dbSNP
rs906020097	1204	dbSNP
rs1000234036	1209	dbSNP
rs904522577	1213	dbSNP
rs1023113788	1215	dbSNP
rs1208183053	1216	dbSNP
rs369820359	1222	dbSNP
rs946275500	1223	dbSNP
rs1261074233	1228	dbSNP
rs773948823	1229	dbSNP
rs1201567750	1230	dbSNP
rs1174772231	1231	dbSNP
rs12818552	1231	dbSNP
rs1458441526	1232	dbSNP
rs933743948	1233	dbSNP
rs899280911	1234	dbSNP
rs920977376	1236	dbSNP
rs1037736616	1237	dbSNP
rs148009304	1239	dbSNP
rs200051704	1239	dbSNP
rs539011209	1239	dbSNP
rs1390427612	1240	dbSNP
rs941069347	1242	dbSNP
rs1396848637	1248	dbSNP
rs1268627554	1249	dbSNP
rs1299370603	1251	dbSNP
rs1490343012	1260	dbSNP
rs1464054077	1262	dbSNP
rs534382893	1267	dbSNP
rs1473125972	1268	dbSNP
rs4237789	1274	dbSNP
rs777576072	1276	dbSNP
rs913771013	1278	dbSNP
rs1362923480	1279	dbSNP
rs1499681	1295	dbSNP
rs1455571004	1299	dbSNP
rs1024146047	1303	dbSNP
rs1378218224	1310	dbSNP
rs114982149	1311	dbSNP
rs958522930	1313	dbSNP
rs1301254854	1315	dbSNP
rs1352031786	1319	dbSNP
rs200753251	1321	dbSNP
rs565626388	1323	dbSNP
rs1034475467	1324	dbSNP
rs1247051266	1325	dbSNP
rs980532627	1325	dbSNP
rs1266057144	1326	dbSNP
rs969095388	1330	dbSNP
rs1485365128	1332	dbSNP
rs1281152941	1335	dbSNP
rs1220817714	1345	dbSNP
rs1000260379	1350	dbSNP
rs1254611003	1350	dbSNP
rs904550018	1355	dbSNP
rs1177059323	1356	dbSNP
rs748036894	1360	dbSNP
rs569227107	1362	dbSNP
rs551005228	1363	dbSNP
rs12302908	1364	dbSNP
rs371757933	1367	dbSNP
rs1161539725	1379	dbSNP
rs1372397843	1381	dbSNP
rs369539586	1382	dbSNP
rs890662603	1396	dbSNP
rs1308611849	1403	dbSNP
rs1051886377	1405	dbSNP
rs899142539	1406	dbSNP
rs143478391	1410	dbSNP
rs754984303	1411	dbSNP
rs900675873	1417	dbSNP
rs1315998079	1418	dbSNP
rs1007612673	1420	dbSNP
rs1039458340	1421	dbSNP
rs1051550401	1425	dbSNP
rs1343550384	1425	dbSNP
rs941079430	1429	dbSNP
rs77518455	1433	dbSNP
rs1484463032	1434	dbSNP
rs201976347	1434	dbSNP
rs560795201	1434	dbSNP
rs766939883	1435	dbSNP
rs1243394544	1438	dbSNP
rs1443471588	1441	dbSNP
rs1181255969	1442	dbSNP
rs1157944833	1444	dbSNP
rs766398199	1446	dbSNP
rs936376526	1447	dbSNP
rs1169692348	1449	dbSNP
rs190986922	1454	dbSNP
rs1337621351	1458	dbSNP
rs187405679	1462	dbSNP
rs992743774	1464	dbSNP
rs1362778938	1465	dbSNP
rs754140055	1469	dbSNP
rs758504463	1471	dbSNP
rs1271513115	1473	dbSNP
rs1204779356	1474	dbSNP
rs969127856	1474	dbSNP
rs1258273396	1475	dbSNP
rs1190231771	1479	dbSNP
rs1487585165	1481	dbSNP
rs958554106	1483	dbSNP
rs1034169649	1487	dbSNP
rs1249299521	1489	dbSNP
rs556503335	1491	dbSNP
rs1261571652	1497	dbSNP
rs761373828	1498	dbSNP
rs1417364439	1503	dbSNP
rs978851621	1504	dbSNP
rs993044576	1508	dbSNP
rs1176942245	1512	dbSNP
rs1405595786	1519	dbSNP
rs750576405	1520	dbSNP
rs774205997	1532	dbSNP
rs1345797829	1535	dbSNP
rs182291394	1539	dbSNP
rs1231042672	1542	dbSNP
rs1333787435	1548	dbSNP
rs1224479745	1550	dbSNP
rs1402998962	1553	dbSNP
rs745819551	1559	dbSNP
rs1321889504	1567	dbSNP
rs1221233602	1568	dbSNP
rs994173644	1571	dbSNP
rs554052052	1586	dbSNP
rs190120882	1587	dbSNP
rs1016226891	1590	dbSNP
rs1030579494	1597	dbSNP
rs553018635	1599	dbSNP
rs1428293745	1602	dbSNP
rs889240059	1603	dbSNP
rs1052276002	1607	dbSNP
rs997390001	1613	dbSNP
rs1409067312	1614	dbSNP
rs892277862	1616	dbSNP
rs1162572530	1621	dbSNP
rs1316369816	1626	dbSNP
rs540460652	1628	dbSNP
rs56245303	1628	dbSNP
rs560214845	1629	dbSNP
rs1230685977	1630	dbSNP
rs1388481282	1633	dbSNP
rs138632625	1638	dbSNP
rs574584603	1641	dbSNP
rs1037811470	1647	dbSNP
rs917701062	1658	dbSNP
rs1418918355	1663	dbSNP
rs1281464590	1669	dbSNP
rs992299092	1671	dbSNP
rs1005304826	1672	dbSNP
rs888161809	1685	dbSNP
rs1268116656	1689	dbSNP
rs1489795712	1694	dbSNP
rs1046805536	1700	dbSNP
rs1183812977	1701	dbSNP
rs1233641571	1705	dbSNP
rs1469771670	1707	dbSNP
rs1157075842	1713	dbSNP
rs929672376	1718	dbSNP
rs916902681	1723	dbSNP
rs1425395120	1724	dbSNP
rs536906378	1729	dbSNP
rs929717595	1731	dbSNP
rs1372607719	1733	dbSNP
rs1057097466	1745	dbSNP
rs1194456589	1746	dbSNP
rs937209759	1748	dbSNP
rs1265702828	1753	dbSNP
rs927127523	1759	dbSNP
rs147125882	1760	dbSNP
rs540326503	1761	dbSNP
rs972224934	1761	dbSNP
rs1440976519	1766	dbSNP
rs1016469366	1772	dbSNP
rs532521229	1782	dbSNP
rs1246153684	1788	dbSNP
rs953368658	1790	dbSNP
rs1473551277	1798	dbSNP
rs1234784938	1799	dbSNP
rs988908858	1804	dbSNP
rs1336829710	1807	dbSNP
rs777118436	1807	dbSNP
rs1269133559	1809	dbSNP
rs998029905	1810	dbSNP
rs1030260573	1811	dbSNP
rs996403607	1816	dbSNP
rs1328194367	1818	dbSNP
rs1169001329	1823	dbSNP
rs1323625989	1827	dbSNP
rs1396175743	1833	dbSNP
rs964892996	1837	dbSNP
rs554645840	1840	dbSNP
rs1006836309	1844	dbSNP
rs186153473	1845	dbSNP
rs1408453255	1847	dbSNP
rs1418866289	1854	dbSNP
rs1229472990	1855	dbSNP
rs1168632611	1856	dbSNP
rs543629564	1864	dbSNP
rs761289864	1868	dbSNP
rs181512585	1869	dbSNP
rs560706081	1874	dbSNP
rs746097307	1879	dbSNP
rs1241281538	1882	dbSNP
rs947731179	1889	dbSNP
rs895495215	1897	dbSNP
rs1373055600	1899	dbSNP
rs1255307043	1909	dbSNP
rs1474611748	1911	dbSNP
rs12301352	1913	dbSNP
rs1435765235	1916	dbSNP
rs1271680543	1919	dbSNP
rs1469940389	1920	dbSNP
rs1335713090	1923	dbSNP
rs937242440	1924	dbSNP
rs569173248	1926	dbSNP
rs1403209695	1930	dbSNP
rs927158635	1932	dbSNP
rs1056110375	1935	dbSNP
rs530686276	1936	dbSNP
rs1224951209	1937	dbSNP
rs1301079042	1941	dbSNP
rs770504060	1944	dbSNP
rs1324002613	1946	dbSNP
rs1233518456	1947	dbSNP
rs1261521510	1954	dbSNP
rs1486875882	1955	dbSNP
rs1205833924	1962	dbSNP
rs188691015	1964	dbSNP
rs1371399824	1965	dbSNP
rs1445826875	1968	dbSNP
rs1305415814	1971	dbSNP
rs1202237861	1976	dbSNP
rs58862482	1977	dbSNP
rs1354224320	1979	dbSNP
rs1480328613	1980	dbSNP
rs1175238539	1983	dbSNP
rs1329805734	1986	dbSNP
rs972674630	1987	dbSNP
rs184008349	1993	dbSNP
rs1373739773	2009	dbSNP
rs913046352	2015	dbSNP
rs56882949	2023	dbSNP
rs541074438	2024	dbSNP
rs909490140	2025	dbSNP
rs1397231716	2030	dbSNP
rs1345855731	2031	dbSNP
rs986331585	2032	dbSNP
rs1468273645	2036	dbSNP
rs954770378	2043	dbSNP
rs573790111	2044	dbSNP
rs1260154944	2045	dbSNP
rs1348676520	2046	dbSNP
rs141847102	2048	dbSNP
rs1225311648	2052	dbSNP
rs974682347	2060	dbSNP
rs1277604077	2061	dbSNP
rs964675817	2067	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.

- 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.