>RHOB|NM_004040|3'UTR
   1 GGGCCGCGCCCGTCGCGCCTGCCCCTGCCGGCACGGCTCCCCCTCCTGGACCAGTCCCCCGCGAGCCCGGAGAAGGGGAG
  81 ACCCGTGTCCCACAAGGACCCCACCGGCCTGCCTGGCATCTGTCTGCTGACGCCTCTGGCTTGCGCCAGGACTTGGCGTG
 161 GGCACCGGGCGCCCCCATCCCAGTGTCTGTGTGCGTCCAGCTGTGTTGCACAGGCCTGGGCTCCCCACTGAGTGCCAAGG
 241 GTCCCCTGAGCATGCTTTTCTGAAGAGCCGGGCCTCAGAGTGTGTGGCTGTGTGTCTGTTCGACTCCCCTCGCCCCATTT
 321 TCACCCCACCCCCGCCTCTGATCCCCGGGGGCGAGATTGGCGCGGGAGTGTGGCCGCGCCCCATCAGATGTTCGCCCTTC
 401 ACCAGCGGGAGCTTGATATCCCTTGTCTGTAACATAGACCCCGGGTACTGCGGGAGGGGAGGGCTGCTGGGGAGGATGGG
 481 GGGATGTTATATAAATATAGATATAATTTTATTTTCGGAGCTAAGATGGTGTTATTTAAGGGTGGTGATGGGTGAGCGCT
 561 CTGGCCCAGGCTGGGCCAGACTCCCGCCCAAGCATGAACAGGACTTGACCATCTTTCCAACCCCTGGGGAAGACATTTGC
 641 AACTGACTTGGGGAGGACACAGCTTCAGCACAGCCTCTCCTGCGGGCCAGCCCGCTGCGAACCCTCCACCAGCTACCGGA
 721 GGGAGGAGGGAGGATGCGCTGTGGGGTTGTTTTTGCCATAAGCGAACTTTGTGCCTGTCCTAGAAGTGAAAATTGTTCAG
 801 TCCAAGAAACTGATGTTATTTGATTTATTTAAAGGCTAAAATTTGTTTTTTTATTCTTTGCACAATTGTTTCATTGTTTG
 881 ACACTTAATGCACTCGTCATTTGCATACGACAGTAGCATTCTGACCACACTTGTACGCTGTAACCTCATCTACTTCTGAT
 961 GTTTTTAAAAAATGACTTTTAACAAGGAGAGGGAAAAGAAACCCACTAAATTTTGCTTTGTTTCCTTGAAGAATGTGGCA
1041 ACACTGTTTTGTGATTTTATTTGTGCAGGTCATGCACACAGTTTTGATAAAGGGCAGTAACAAGTATTGGGGCCTATTTT
1121 TTTTTTTTCCACAAGGCATTCTCTAAAGCTATGTGAAATTTTCTCTGCACCTCTGTACAGAGAATACACCTGCCCCTGTA
1201 TATCCTTTTTTCCCCTCCCCTCCCTCCCAGTGGTACTTCTACTAAATTGTTGTCTTGTTTTTTATTTTTTAAATAAACTG
1281 ACAAATGACAAAATGGTGAGCTTATGATGTTTACATAAAAGTTCTATAAGCTGTGTATACAGTTTTTTATGTAAAATATT
1361 AAAAGACTATGATGATGACATTTATAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uccggguucuaguUGUUAACAg 5' |||||||| Target 5' ttttattctttgcACAATTGTt 3'	849 - 870	145.00	-7.10
2	miRNA 3' ucCGGGUUC-UAGUUGUUAACAg 5' |:|| || | ::| |||||| Target 5' ctGTCCTAGAAGTGAAAATTGTt 3'	775 - 797	132.00	-10.20
3	miRNA 3' ucCGGGUUC--UA-GU-UGUUAACAg 5' |: ||:| || || |||:|| | Target 5' ttGTGCAGGTCATGCACACAGTTTTg 3'	1061 - 1086	110.00	-7.72

Mutant ID	Mutant Position	Mutant Source
COSN15942354	5	COSMIC
COSN30484461	30	COSMIC
COSN26649768	32	COSMIC
COSN30462870	41	COSMIC
COSN15626129	57	COSMIC
COSN8277913	85	COSMIC
COSN30189782	86	COSMIC
COSN30137847	161	COSMIC
COSN30136078	206	COSMIC
COSN9872127	221	COSMIC
COSN24299338	297	COSMIC
COSN30172917	337	COSMIC
COSN30536633	339	COSMIC
COSN30139707	353	COSMIC
COSN31534430	379	COSMIC
COSN30530561	502	COSMIC
COSN26554431	571	COSMIC
COSN31486622	680	COSMIC
COSN30139523	720	COSMIC
COSN31552610	762	COSMIC
COSN31568097	813	COSMIC
COSN30510614	878	COSMIC
COSN20096368	1129	COSMIC
COSN30128424	1129	COSMIC
COSN30137378	1151	COSMIC
COSN30143117	1152	COSMIC
COSN21178384	1162	COSMIC
COSN30120026	1194	COSMIC
COSN20096374	1252	COSMIC
COSN30111359	1274	COSMIC
COSN28826170	1362	COSMIC
COSN28677732	1370	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs747686313	2	dbSNP
rs769391783	3	dbSNP
rs902867665	6	dbSNP
rs1027591353	7	dbSNP
rs773007113	9	dbSNP
rs1419886688	10	dbSNP
rs762641332	12	dbSNP
rs770690133	13	dbSNP
rs1465534024	14	dbSNP
rs1056788273	15	dbSNP
rs1376288063	17	dbSNP
rs774077364	18	dbSNP
rs1173737118	22	dbSNP
rs1185365126	22	dbSNP
rs950323657	23	dbSNP
rs1456289638	24	dbSNP
rs536929824	25	dbSNP
rs1390321285	27	dbSNP
rs752630835	30	dbSNP
rs1323593106	31	dbSNP
rs1012671009	32	dbSNP
rs1016446156	34	dbSNP
rs765233883	35	dbSNP
rs758687374	36	dbSNP
rs1283923033	40	dbSNP
rs1341331781	42	dbSNP
rs993765876	43	dbSNP
rs1062192	44	dbSNP
rs755323520	46	dbSNP
rs980288357	48	dbSNP
rs765498047	49	dbSNP
rs1209484145	50	dbSNP
rs748602850	51	dbSNP
rs769446969	52	dbSNP
rs777479853	53	dbSNP
rs1194801069	55	dbSNP
rs1162133084	57	dbSNP
rs989654946	61	dbSNP
rs954969552	62	dbSNP
rs552741297	63	dbSNP
rs1172033003	64	dbSNP
rs1427112454	66	dbSNP
rs1261397545	75	dbSNP
rs987899252	76	dbSNP
rs1017601191	79	dbSNP
rs1490742881	83	dbSNP
rs1251266220	85	dbSNP
rs964798914	86	dbSNP
rs1270095137	92	dbSNP
rs945667465	93	dbSNP
rs1260274467	96	dbSNP
rs989490046	97	dbSNP
rs1433011797	106	dbSNP
rs1197177858	107	dbSNP
rs1375349744	109	dbSNP
rs911903223	114	dbSNP
rs1168047909	119	dbSNP
rs1439560835	119	dbSNP
rs1400304384	120	dbSNP
rs914409574	125	dbSNP
rs572788690	127	dbSNP
rs1426820400	128	dbSNP
rs977379701	129	dbSNP
rs1390258182	132	dbSNP
rs924553263	147	dbSNP
rs1456006736	151	dbSNP
rs938595473	153	dbSNP
rs1384920766	164	dbSNP
rs1393835067	170	dbSNP
rs1193519292	172	dbSNP
rs1458276469	172	dbSNP
rs543503259	173	dbSNP
rs894135983	177	dbSNP
rs1326959232	178	dbSNP
rs1250597906	180	dbSNP
rs182696067	184	dbSNP
rs1206640127	188	dbSNP
rs1349733627	188	dbSNP
rs1037430477	196	dbSNP
rs766186117	198	dbSNP
rs993694109	199	dbSNP
rs575384054	200	dbSNP
rs886347990	201	dbSNP
rs1026463363	202	dbSNP
rs544438089	204	dbSNP
rs1264226469	210	dbSNP
rs561051179	212	dbSNP
rs1023962119	215	dbSNP
rs1373959207	217	dbSNP
rs1448100869	230	dbSNP
rs1009143766	235	dbSNP
rs1440834367	240	dbSNP
rs1365678681	243	dbSNP
rs906877586	244	dbSNP
rs1001270444	246	dbSNP
rs1034519975	255	dbSNP
rs959499684	256	dbSNP
rs529987335	259	dbSNP
rs1277903211	264	dbSNP
rs1018029864	269	dbSNP
rs540508245	270	dbSNP
rs989601037	275	dbSNP
rs1351267412	277	dbSNP
rs1022456458	281	dbSNP
rs62121967	283	dbSNP
rs1164278548	289	dbSNP
rs989543579	289	dbSNP
rs569827161	293	dbSNP
rs911865998	296	dbSNP
rs1312649637	297	dbSNP
rs528486957	297	dbSNP
rs966754170	299	dbSNP
rs751476861	302	dbSNP
rs551332362	307	dbSNP
rs112622561	308	dbSNP
rs922017034	310	dbSNP
rs1474345310	314	dbSNP
rs1255183244	315	dbSNP
rs1246153963	318	dbSNP
rs1313735587	322	dbSNP
rs1022788428	324	dbSNP
rs1186927878	326	dbSNP
rs952554781	329	dbSNP
rs1262895906	330	dbSNP
rs1486419258	331	dbSNP
rs1286302783	332	dbSNP
rs1203244934	333	dbSNP
rs992717719	339	dbSNP
rs1234826755	344	dbSNP
rs1048908120	345	dbSNP
rs1293112210	348	dbSNP
rs915594946	353	dbSNP
rs1328497004	355	dbSNP
rs886303721	356	dbSNP
rs1382141444	357	dbSNP
rs530897155	367	dbSNP
rs1391623300	368	dbSNP
rs948347009	371	dbSNP
rs1184288998	374	dbSNP
rs1037354480	375	dbSNP
rs920372474	376	dbSNP
rs1046079297	381	dbSNP
rs1475624052	382	dbSNP
rs929063323	383	dbSNP
rs1191010832	384	dbSNP
rs1048401573	394	dbSNP
rs1062292	395	dbSNP
rs1001618535	406	dbSNP
rs567846448	407	dbSNP
rs536584682	408	dbSNP
rs892886452	410	dbSNP
rs1039258105	412	dbSNP
rs1214732513	413	dbSNP
rs1022405809	414	dbSNP
rs966860535	419	dbSNP
rs900528069	420	dbSNP
rs1227391330	421	dbSNP
rs553639534	422	dbSNP
rs1393749586	423	dbSNP
rs567008393	425	dbSNP
rs966798421	429	dbSNP
rs1165911058	432	dbSNP
rs187327344	434	dbSNP
rs1395639115	435	dbSNP
rs1031922835	436	dbSNP
rs546625861	438	dbSNP
rs952096790	447	dbSNP
rs1251818066	449	dbSNP
rs993174643	450	dbSNP
rs915856379	455	dbSNP
rs1439160438	463	dbSNP
rs1271383027	466	dbSNP
rs1195605218	469	dbSNP
rs1348995261	471	dbSNP
rs748722763	474	dbSNP
rs1370420337	478	dbSNP
rs1228236654	483	dbSNP
rs1170526386	486	dbSNP
rs1266553829	489	dbSNP
rs558555999	491	dbSNP
rs1328381187	495	dbSNP
rs780074959	495	dbSNP
rs1255521192	499	dbSNP
rs1452494365	501	dbSNP
rs928537631	501	dbSNP
rs1482342038	517	dbSNP
rs1179004962	521	dbSNP
rs1470849345	523	dbSNP
rs1230849120	526	dbSNP
rs1251591048	527	dbSNP
rs1472386619	530	dbSNP
rs1282899407	531	dbSNP
rs1179758040	540	dbSNP
rs1347754283	542	dbSNP
rs192230103	544	dbSNP
rs929051242	546	dbSNP
rs1055849317	548	dbSNP
rs1047439620	549	dbSNP
rs892831018	557	dbSNP
rs1011250287	558	dbSNP
rs1470278522	564	dbSNP
rs912059391	570	dbSNP
rs944868280	572	dbSNP
rs1358524087	585	dbSNP
rs1039158786	587	dbSNP
rs1357334628	589	dbSNP
rs78953415	590	dbSNP
rs1011104366	596	dbSNP
rs777635201	603	dbSNP
rs1475330353	608	dbSNP
rs183777378	610	dbSNP
rs902481703	612	dbSNP
rs1000033993	613	dbSNP
rs1407552048	614	dbSNP
rs1485521389	621	dbSNP
rs1243233726	624	dbSNP
rs1035089802	632	dbSNP
rs961059642	649	dbSNP
rs1234174622	655	dbSNP
rs574581714	659	dbSNP
rs1014119185	663	dbSNP
rs1291610643	665	dbSNP
rs1412732608	666	dbSNP
rs1347730085	667	dbSNP
rs865958995	672	dbSNP
rs1289390871	674	dbSNP
rs1022798166	675	dbSNP
rs1028970292	676	dbSNP
rs1343000284	680	dbSNP
rs1157739248	684	dbSNP
rs1220149044	685	dbSNP
rs1278600324	688	dbSNP
rs952044674	691	dbSNP
rs984834809	698	dbSNP
rs115786444	699	dbSNP
rs1206800651	700	dbSNP
rs1251897372	701	dbSNP
rs973277801	702	dbSNP
rs962187784	703	dbSNP
rs560416281	704	dbSNP
rs981681577	706	dbSNP
rs928513101	712	dbSNP
rs1469844739	713	dbSNP
rs1271510864	718	dbSNP
rs747795887	718	dbSNP
rs950384112	718	dbSNP
rs983624462	726	dbSNP
rs1216302443	728	dbSNP
rs937240026	738	dbSNP
rs1055649112	741	dbSNP
rs1441045124	746	dbSNP
rs1203232740	749	dbSNP
rs1323498084	755	dbSNP
rs1457090354	757	dbSNP
rs1355520601	758	dbSNP
rs1172959302	759	dbSNP
rs1427055126	772	dbSNP
rs756811744	778	dbSNP
rs944439429	780	dbSNP
rs532271403	781	dbSNP
rs527953953	783	dbSNP
rs1190395527	785	dbSNP
rs1481066796	789	dbSNP
rs780381797	790	dbSNP
rs1423055750	792	dbSNP
rs1305510406	793	dbSNP
rs1008016228	795	dbSNP
rs922099372	798	dbSNP
rs189132194	799	dbSNP
rs1043919816	800	dbSNP
rs1300585371	802	dbSNP
rs902533337	803	dbSNP
rs760529272	804	dbSNP
rs1369349798	805	dbSNP
rs564914120	808	dbSNP
rs1385921221	811	dbSNP
rs1427458906	817	dbSNP
rs1304626355	818	dbSNP
rs1365789240	819	dbSNP
rs1056856754	820	dbSNP
rs996808288	822	dbSNP
rs1029252275	823	dbSNP
rs1437706483	839	dbSNP
rs887744686	842	dbSNP
rs1437123211	843	dbSNP
rs1428952700	845	dbSNP
rs1198227664	846	dbSNP
rs546526980	846	dbSNP
rs1419702977	857	dbSNP
rs896604900	861	dbSNP
rs1012336695	865	dbSNP
rs1366123245	866	dbSNP
rs1211557802	869	dbSNP
rs1022808054	873	dbSNP
rs771292400	874	dbSNP
rs530959398	877	dbSNP
rs1278038121	884	dbSNP
rs752879312	885	dbSNP
rs905661647	888	dbSNP
rs550651135	889	dbSNP
rs1298457112	892	dbSNP
rs1389305457	894	dbSNP
rs1350328288	897	dbSNP
rs1313839160	900	dbSNP
rs1417221267	909	dbSNP
rs192183115	911	dbSNP
rs1172465345	924	dbSNP
rs994769680	926	dbSNP
rs1294292360	929	dbSNP
rs1452398811	932	dbSNP
rs962422029	933	dbSNP
rs1027548784	935	dbSNP
rs981576363	937	dbSNP
rs530319469	938	dbSNP
rs1188417799	939	dbSNP
rs1486506691	941	dbSNP
rs1253998248	945	dbSNP
rs958582340	947	dbSNP
rs1350831449	948	dbSNP
rs1261965628	949	dbSNP
rs1328027616	951	dbSNP
rs1293479512	957	dbSNP
rs1186995468	961	dbSNP
rs950673536	962	dbSNP
rs555423149	967	dbSNP
rs747569087	973	dbSNP
rs1456727616	977	dbSNP
rs145967445	977	dbSNP
rs1203391587	980	dbSNP
rs1385553354	984	dbSNP
rs779799104	985	dbSNP
rs1476071024	992	dbSNP
rs1366481649	995	dbSNP
rs1186830813	996	dbSNP
rs1447484234	999	dbSNP
rs1019016733	1004	dbSNP
rs1263286838	1005	dbSNP
rs947339310	1007	dbSNP
rs79242034	1008	dbSNP
rs924324066	1013	dbSNP
rs139892391	1017	dbSNP
rs769396459	1017	dbSNP
rs943757156	1021	dbSNP
rs1040817653	1025	dbSNP
rs1328729375	1028	dbSNP
rs373830882	1029	dbSNP
rs946864998	1035	dbSNP
rs1395231472	1040	dbSNP
rs1050618557	1042	dbSNP
rs1328236838	1043	dbSNP
rs1348454769	1045	dbSNP
rs1460066494	1047	dbSNP
rs1388355587	1050	dbSNP
rs1433283375	1058	dbSNP
rs769212673	1064	dbSNP
rs1374758338	1067	dbSNP
rs924247250	1070	dbSNP
rs935575357	1072	dbSNP
rs776978328	1078	dbSNP
rs1253066902	1080	dbSNP
rs1215252428	1089	dbSNP
rs1288639612	1093	dbSNP
rs1449486373	1093	dbSNP
rs1196775205	1094	dbSNP
rs552232208	1097	dbSNP
rs568858085	1101	dbSNP
rs1341826548	1102	dbSNP
rs896439738	1105	dbSNP
rs1226359339	1106	dbSNP
rs1014923532	1107	dbSNP
rs1357336337	1108	dbSNP
rs1371900302	1113	dbSNP
rs1305679156	1115	dbSNP
rs537797427	1116	dbSNP
rs1166705204	1117	dbSNP
rs1398290385	1117	dbSNP
rs1410332125	1117	dbSNP
rs1429024839	1117	dbSNP
rs34358430	1117	dbSNP
rs1227907720	1118	dbSNP
rs1045144765	1122	dbSNP
rs1476732075	1129	dbSNP
rs397763503	1129	dbSNP
rs79591078	1129	dbSNP
rs1287508076	1130	dbSNP
rs62121968	1131	dbSNP
rs1318545552	1137	dbSNP
rs1439974019	1139	dbSNP
rs905712709	1141	dbSNP
rs1003344030	1144	dbSNP
rs77112597	1145	dbSNP
rs958748032	1150	dbSNP
rs1027887431	1163	dbSNP
rs886391659	1166	dbSNP
rs1217579317	1168	dbSNP
rs1184910270	1170	dbSNP
rs1005204151	1171	dbSNP
rs184500234	1172	dbSNP
rs1352176791	1180	dbSNP
rs1472276511	1181	dbSNP
rs1161133569	1185	dbSNP
rs548089709	1188	dbSNP
rs995892686	1190	dbSNP
rs977337322	1193	dbSNP
rs1402863687	1195	dbSNP
rs1160359796	1198	dbSNP
rs868199028	1199	dbSNP
rs924249171	1205	dbSNP
rs943721227	1206	dbSNP
rs1250122772	1207	dbSNP
rs553668877	1209	dbSNP
rs1028725833	1211	dbSNP
rs1260238609	1211	dbSNP
rs1208574121	1212	dbSNP
rs1311738043	1212	dbSNP
rs1285043478	1215	dbSNP
rs576755362	1219	dbSNP
rs1327318494	1220	dbSNP
rs1287917619	1222	dbSNP
rs1227557029	1230	dbSNP
rs1355786413	1231	dbSNP
rs1304568497	1234	dbSNP
rs976875518	1236	dbSNP
rs1433454556	1238	dbSNP
rs967760326	1241	dbSNP
rs1318380146	1242	dbSNP
rs56774510	1247	dbSNP
rs71820361	1247	dbSNP
rs1157872892	1250	dbSNP
rs932430876	1250	dbSNP
rs1050567937	1252	dbSNP
rs1282693896	1255	dbSNP
rs1420457666	1255	dbSNP
rs1483193246	1255	dbSNP
rs887976267	1255	dbSNP
rs766195247	1258	dbSNP
rs942229166	1258	dbSNP
rs979606834	1266	dbSNP
rs1002882570	1269	dbSNP
rs752153393	1270	dbSNP
rs1226583956	1273	dbSNP
rs1378375872	1279	dbSNP
rs1327018184	1281	dbSNP
rs769983217	1285	dbSNP
rs894560724	1291	dbSNP
rs1012977406	1294	dbSNP
rs1021400593	1296	dbSNP
rs956981145	1297	dbSNP
rs992787020	1301	dbSNP
rs968540787	1302	dbSNP
rs1278754702	1303	dbSNP
rs998689757	1304	dbSNP
rs1423553594	1305	dbSNP
rs1031596333	1308	dbSNP
rs965710269	1311	dbSNP
rs1447792670	1315	dbSNP
rs1247480989	1323	dbSNP
rs976443542	1324	dbSNP
rs1469975477	1325	dbSNP
rs773507358	1327	dbSNP
rs1320824380	1331	dbSNP
rs143778278	1337	dbSNP
rs1230505873	1338	dbSNP
rs953765912	1339	dbSNP
rs909354932	1343	dbSNP
rs983887066	1343	dbSNP
rs766485608	1344	dbSNP
rs1291392429	1350	dbSNP
rs756915305	1361	dbSNP
rs1355461640	1363	dbSNP
rs920035516	1366	dbSNP
rs1179247745	1368	dbSNP
rs564979038	1368	dbSNP
rs558100385	1369	dbSNP
rs1434938773	1370	dbSNP
rs144805732	1373	dbSNP
rs1048924623	1377	dbSNP
rs1190771709	1377	dbSNP
rs1413989139	1379	dbSNP
rs3180120	1379	dbSNP
rs1233239215	1380	dbSNP
rs1488069190	1380	dbSNP
rs3180121	1381	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.

- 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.