>DAZAP2|NM_001136264|3'UTR
   1 GGAACCAAGGCCACCTCTGTGCCGGGAAAGACATCACATACCTTCAGCACTTCTCACAATGTAACTGCTTTAGTCATATT
  81 AACCTGAAGTTGCAGTTTAGACACATGTTGTTGGGGTGTCTTTCTGGTGCCCAAACTTTCAGGCACTTTTCAAATTTAAT
 161 AAGGAACCATGTAATGGTAGCAGTACCTCCCTAAAGCATTTTGAGGTAGGGGAGGTATCCATTCATAAAATGAATGTGGG
 241 TGAAGCCGCCCTAAGGATTTTCCTTTAATTTCTCTGGAGTAATACTGTACCATACTGGTCTTTGCTTTTAGTAATAAAAC
 321 ATCAAATTAGGTTTGGAGGGAACTTTGATCTTCCTAAGAATTAAAGTTGCCAAATTATTCTGATTGGTCTTTAATCTCCT
 401 TTAAGTCTTTGATATATATTACTTGTTATAAATGGAACGCATTAGTTGTCTGCCTTTTCCTTTCCATCCCTTGCCCCACC
 481 CATCCCATCTCCAACCCTAGTCTTCCATTTCCTCCCGCCAGTCTCCATTGAATCAATGGTGCAGGACAGAAAGCCAGTCA
 561 GACTAATTTCCTTCTTTCCTCGCACTTCTCCCCACTCGTCATCTTTTAACTAGTGTTTCACAAGGATCCTCTGAAACCCT
 641 CTCTGTGCCCCAAGTACAGATGCCATTACTTCTGCTTTCGTATCTCCTCAGGCAAAAGTGGAGGGTGCCTTATGGGCCCT
 721 CCTCATAGGTTGTCTCTGCATACACGAACCTAACCCAAATTTGCTTTGGTGCCAGAAAAACTGAGCTATGTTTGAACAAA
 801 GATGTCGTGCAAACTGTACTGTGAACAACAGTTGGTTTAAAATATGAGGGGCAAGGAGGAGGATGCATTTCAAAAGCTTG
 881 ATTGATGTGTTCAGAGCTAAATTAAGAGGAGTTTTCAGATCAAAAACTGGTTACCATTTTTTGTCAGAGTGTCTGATGCG
 961 GCCACTCATTCGGCTCCCCAGAATTCCTAGACTGGGTTAATAGGGTCATATTGTGAATGTCTCACTACAAAATGACTTGA
1041 GTCCAGTGAAATCTCATTAGGGTTTAAGAATATTTCAGGGATCCTTAATGTTTTGATTTTTGTTTTCTGAAATTGGATTT
1121 TATTTTATTTTATCTTATAATTTCAGTTCATCTAAATTGTGTGTTCTGTACATGTGATGTTTGACTGTACCATTGACTGT
1201 TATGGAAGTTCAGCGTTGTATGTCTCTCTCTACACTGTGGTGCACTTAACTTGTGGAATTTTTATACTAAAAATGTAGAA
1281 TAAAGACTATTTTGAAGATTTGAATAAAGTGATGAAGTTGCATTACACCTCACTGCAAGGATTCTTTACTTAGCTTGTTT
1361 TTAGATTTCTTCTATATATATTTTATTTATATCCCATCTAGAATTCAGCTAGGTGCTGCTGCTGCTCTGTTTCCTTTGAT
1441 GACGCTTTGAAATAAAGGCAGGAGTACAAGCCTAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uaGGAGAUUU--UUCUACGUGAAa 5' :||||| | : |:||||||| Target 5' tcTCTCTACACTGTGGTGCACTTa 3'	1225 - 1248	162.00	-15.70
2	miRNA 3' uaggagaUUUUUCUACGUGAAa 5' |::|:||||||:|| Target 5' ggcaaggAGGAGGATGCATTTc 3'	850 - 871	147.00	-13.80
3	miRNA 3' uaggaGAUU---UUUCUACGUGAAa 5' :||| :||| |||| || Target 5' tcataTTAACCTGAAGTTGCAGTTt 3'	74 - 98	125.00	-9.10

Mutant ID	Mutant Position	Mutant Source
COSM8179832	11	COSMIC
COSM2009239	25	COSMIC
COSM6640143	32	COSMIC
COSM2009240	46	COSMIC
COSM8638713	54	COSMIC
COSM7639144	76	COSMIC
COSM8719256	101	COSMIC
COSM9112114	111	COSMIC
COSM9834883	111	COSMIC
COSM4879998	125	COSMIC
COSM9834884	131	COSMIC
COSM9579709	141	COSMIC
COSM9834885	141	COSMIC
COSM4042675	148	COSMIC
COSM9857964	152	COSMIC
COSM1739875	166	COSMIC
COSM9834886	176	COSMIC
COSM9834887	185	COSMIC
COSN30160074	225	COSMIC
COSN31515730	273	COSMIC
COSN30526686	315	COSMIC
COSN31485493	343	COSMIC
COSN31548028	412	COSMIC
COSN28722043	471	COSMIC
COSN28798880	479	COSMIC
COSN30542021	518	COSMIC
COSN24470830	663	COSMIC
COSN26649497	795	COSMIC
COSN20722565	972	COSMIC
COSN31570941	1049	COSMIC
COSN21848396	1122	COSMIC
COSN31609023	1271	COSMIC
COSN31515538	1273	COSMIC
COSN5834608	1279	COSMIC
COSN31568582	1370	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs377252029	6	dbSNP
rs1444879102	11	dbSNP
rs1321523231	12	dbSNP
rs779908029	14	dbSNP
rs1049467	18	dbSNP
rs780268345	20	dbSNP
rs749383352	21	dbSNP
rs1356278307	22	dbSNP
rs866151968	23	dbSNP
rs201854240	24	dbSNP
rs374168708	25	dbSNP
rs748645128	26	dbSNP
rs772739085	43	dbSNP
rs1273115172	45	dbSNP
rs1237312012	47	dbSNP
rs1442851717	51	dbSNP
rs773665805	54	dbSNP
rs1388517124	56	dbSNP
rs373137618	58	dbSNP
rs776209061	59	dbSNP
rs1194425333	60	dbSNP
rs759069912	62	dbSNP
rs1246428707	63	dbSNP
rs764700099	69	dbSNP
rs1453823665	76	dbSNP
rs752647176	76	dbSNP
rs758289456	79	dbSNP
rs1416463152	80	dbSNP
rs190665763	81	dbSNP
rs764040405	85	dbSNP
rs1340353423	89	dbSNP
rs1401170814	95	dbSNP
rs1278257723	101	dbSNP
rs751297697	104	dbSNP
rs746969932	106	dbSNP
rs1170336145	109	dbSNP
rs757111801	121	dbSNP
rs780242085	124	dbSNP
rs768400345	127	dbSNP
rs749523180	135	dbSNP
rs1485275086	137	dbSNP
rs1194362350	139	dbSNP
rs746420669	141	dbSNP
rs1299393933	147	dbSNP
rs1388262116	148	dbSNP
rs755057505	150	dbSNP
rs1476920867	153	dbSNP
rs1272850169	160	dbSNP
rs987071954	162	dbSNP
rs1346044700	170	dbSNP
rs377433711	174	dbSNP
rs1479147558	180	dbSNP
rs1171139324	182	dbSNP
rs1402007396	183	dbSNP
rs776378700	189	dbSNP
rs1403859391	194	dbSNP
rs1300666893	196	dbSNP
rs1361876745	197	dbSNP
rs1410677356	199	dbSNP
rs1015602736	206	dbSNP
rs1481731111	209	dbSNP
rs748284553	211	dbSNP
rs1182027233	215	dbSNP
rs1331284058	222	dbSNP
rs1234985252	226	dbSNP
rs567234272	228	dbSNP
rs1252488156	233	dbSNP
rs1272833304	240	dbSNP
rs1346932287	242	dbSNP
rs948850857	247	dbSNP
rs980314694	248	dbSNP
rs536198616	249	dbSNP
rs1345866857	250	dbSNP
rs980592380	254	dbSNP
rs1407852894	255	dbSNP
rs775859943	272	dbSNP
rs1348711188	274	dbSNP
rs938558274	281	dbSNP
rs14678	286	dbSNP
rs1197419482	290	dbSNP
rs1443268477	291	dbSNP
rs760873231	292	dbSNP
rs1358771802	294	dbSNP
rs1174171357	306	dbSNP
rs1471176274	310	dbSNP
rs552716848	330	dbSNP
rs1430351838	346	dbSNP
rs1159084577	348	dbSNP
rs182906473	349	dbSNP
rs1230705337	353	dbSNP
rs1052999201	354	dbSNP
rs891448299	355	dbSNP
rs947076145	373	dbSNP
rs1205119086	374	dbSNP
rs1235409481	374	dbSNP
rs1470930155	379	dbSNP
rs574069333	385	dbSNP
rs544746793	388	dbSNP
rs1042721105	401	dbSNP
rs1480309011	406	dbSNP
rs1331107409	413	dbSNP
rs572861649	415	dbSNP
rs1309294015	420	dbSNP
rs1224436388	423	dbSNP
rs1378467942	426	dbSNP
rs1174790553	430	dbSNP
rs187567108	439	dbSNP
rs900219048	440	dbSNP
rs1408902235	455	dbSNP
rs562967636	460	dbSNP
rs1050309711	461	dbSNP
rs1160412962	466	dbSNP
rs1422746372	467	dbSNP
rs1328211840	470	dbSNP
rs15839	477	dbSNP
rs1185053661	478	dbSNP
rs891331325	490	dbSNP
rs578210065	498	dbSNP
rs1206556534	504	dbSNP
rs1486701669	505	dbSNP
rs1352851703	512	dbSNP
rs1243904816	513	dbSNP
rs1444548722	517	dbSNP
rs1008890435	518	dbSNP
rs1248520715	522	dbSNP
rs138200161	529	dbSNP
rs190893016	536	dbSNP
rs1297373967	540	dbSNP
rs1430864303	549	dbSNP
rs1015885498	555	dbSNP
rs763165216	556	dbSNP
rs1284997619	560	dbSNP
rs961351065	567	dbSNP
rs995483692	574	dbSNP
rs1347677655	579	dbSNP
rs1164919195	580	dbSNP
rs1027001264	582	dbSNP
rs1389202667	583	dbSNP
rs970512665	586	dbSNP
rs1217957754	589	dbSNP
rs1430098040	591	dbSNP
rs1270197430	594	dbSNP
rs1209240704	595	dbSNP
rs764393840	598	dbSNP
rs1204677610	599	dbSNP
rs1224774585	601	dbSNP
rs1360202558	611	dbSNP
rs1245752148	615	dbSNP
rs1274214712	621	dbSNP
rs1328567899	622	dbSNP
rs1296734389	624	dbSNP
rs980213684	627	dbSNP
rs1489745529	629	dbSNP
rs183437083	637	dbSNP
rs542971167	644	dbSNP
rs1344140153	646	dbSNP
rs928450366	648	dbSNP
rs1424773431	651	dbSNP
rs1130598	663	dbSNP
rs1431933351	666	dbSNP
rs1463198170	671	dbSNP
rs988669890	680	dbSNP
rs762092834	681	dbSNP
rs79990195	683	dbSNP
rs1255610504	685	dbSNP
rs551460767	686	dbSNP
rs1418071214	687	dbSNP
rs1270997156	688	dbSNP
rs112397706	697	dbSNP
rs1199995136	699	dbSNP
rs1411715820	706	dbSNP
rs1490536903	708	dbSNP
rs1273924919	712	dbSNP
rs1329677796	715	dbSNP
rs1365155020	718	dbSNP
rs776926358	720	dbSNP
rs1194361749	726	dbSNP
rs1403366797	727	dbSNP
rs947317953	737	dbSNP
rs1252304971	741	dbSNP
rs1042667390	746	dbSNP
rs1285769522	747	dbSNP
rs1406017424	749	dbSNP
rs921614168	752	dbSNP
rs1326268594	759	dbSNP
rs1396734907	763	dbSNP
rs113479053	768	dbSNP
rs1399246747	791	dbSNP
rs1051557904	799	dbSNP
rs891278165	804	dbSNP
rs560246340	807	dbSNP
rs530580240	808	dbSNP
rs1179720051	813	dbSNP
rs1439816584	819	dbSNP
rs1251545836	826	dbSNP
rs897383397	828	dbSNP
rs117882905	830	dbSNP
rs187326882	836	dbSNP
rs1224729728	843	dbSNP
rs1329057200	845	dbSNP
rs1356764592	846	dbSNP
rs1240595595	847	dbSNP
rs1224270935	852	dbSNP
rs1271414957	853	dbSNP
rs1335780973	859	dbSNP
rs754989656	874	dbSNP
rs970123863	875	dbSNP
rs1449924578	878	dbSNP
rs1002012634	883	dbSNP
rs567524207	885	dbSNP
rs1408112303	889	dbSNP
rs1035803032	891	dbSNP
rs1240840085	899	dbSNP
rs536171824	911	dbSNP
rs1168523635	912	dbSNP
rs1160179730	915	dbSNP
rs1384706554	922	dbSNP
rs10876156	928	dbSNP
rs1364389055	937	dbSNP
rs1165704261	946	dbSNP
rs1460142883	946	dbSNP
rs1245412490	947	dbSNP
rs1204681435	951	dbSNP
rs752549380	960	dbSNP
rs1282793081	965	dbSNP
rs1207349177	972	dbSNP
rs1399875327	973	dbSNP
rs1316015322	980	dbSNP
rs1283080852	982	dbSNP
rs1804087	984	dbSNP
rs1431422654	987	dbSNP
rs538667949	990	dbSNP
rs1130641	1000	dbSNP
rs1379950691	1002	dbSNP
rs531183779	1006	dbSNP
rs921585425	1011	dbSNP
rs1434131204	1012	dbSNP
rs931698243	1021	dbSNP
rs1320211543	1024	dbSNP
rs1401214806	1029	dbSNP
rs1385839952	1030	dbSNP
rs1162989967	1039	dbSNP
rs1051936014	1040	dbSNP
rs911623900	1046	dbSNP
rs1184387238	1051	dbSNP
rs940437003	1051	dbSNP
rs11555478	1086	dbSNP
rs1240652714	1089	dbSNP
rs1196035703	1090	dbSNP
rs1448067857	1091	dbSNP
rs146146716	1117	dbSNP
rs373390567	1117	dbSNP
rs1219579401	1123	dbSNP
rs570998465	1123	dbSNP
rs750889518	1123	dbSNP
rs192230455	1124	dbSNP
rs1048669387	1128	dbSNP
rs749397615	1130	dbSNP
rs1271702654	1137	dbSNP
rs1431458998	1139	dbSNP
rs1194529492	1142	dbSNP
rs1242538893	1153	dbSNP
rs1001518070	1159	dbSNP
rs1367462800	1161	dbSNP
rs1164454595	1164	dbSNP
rs1409599023	1167	dbSNP
rs537613123	1177	dbSNP
rs1417881988	1179	dbSNP
rs1169983840	1181	dbSNP
rs1442848132	1182	dbSNP
rs1476050084	1187	dbSNP
rs1036112856	1194	dbSNP
rs551711588	1195	dbSNP
rs1490699116	1202	dbSNP
rs1185554208	1203	dbSNP
rs895869830	1204	dbSNP
rs1305381743	1212	dbSNP
rs1010278173	1215	dbSNP
rs557601567	1216	dbSNP
rs968553922	1223	dbSNP
rs1156543080	1229	dbSNP
rs1386888416	1230	dbSNP
rs1207090549	1231	dbSNP
rs1401152198	1240	dbSNP
rs1406620727	1244	dbSNP
rs1335999183	1246	dbSNP
rs1303820003	1264	dbSNP
rs1441815651	1266	dbSNP
rs978637806	1276	dbSNP
rs1403889624	1288	dbSNP
rs1140398	1306	dbSNP
rs1410900869	1322	dbSNP
rs751449021	1323	dbSNP
rs777618382	1341	dbSNP
rs1481697995	1361	dbSNP
rs987127853	1363	dbSNP
rs1176096790	1367	dbSNP
rs1035517058	1381	dbSNP
rs895691390	1383	dbSNP
rs1013063583	1385	dbSNP
rs1236452826	1387	dbSNP
rs746678770	1389	dbSNP
rs1460707075	1390	dbSNP
rs1261015229	1392	dbSNP
rs184668320	1399	dbSNP
rs971846387	1410	dbSNP
rs968886784	1414	dbSNP
rs931491960	1417	dbSNP
rs1371211431	1422	dbSNP
rs1300126399	1423	dbSNP
rs1443200037	1425	dbSNP
rs76567392	1425	dbSNP
rs1335118179	1427	dbSNP
rs1471267859	1435	dbSNP
rs1049057269	1436	dbSNP
rs999794608	1443	dbSNP
rs937596680	1444	dbSNP
rs1460405492	1450	dbSNP
rs1205602271	1452	dbSNP
rs1177490306	1464	dbSNP
rs141787286	1465	dbSNP
rs541596133	1469	dbSNP
rs1205294723	1470	dbSNP
rs150498248	1472	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.

- 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.