ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cacAGAUAUGAGACGGUGAAAu 5' ||| ||: | |:||||| Target 5' tctTCTTTATAATTCTACTTTg 3'	145 - 166	143.00	-7.40
2	miRNA 3' caCAGAUAUGAGACGGUGAAAu 5' | :||||| || |||||| Target 5' atGCTTATACACTTACACTTTa 3'	81 - 102	140.00	-13.40
3	miRNA 3' cacAGAUAUGAGAC-----GGUGAAAu 5' || | |:|||| :::|||| Target 5' aatTCCAAATTCTGCTTGCTTGCTTTt 3'	44 - 70	111.00	-8.80

Mutant ID	Mutant Position	Mutant Source
COSN29513540	3	COSMIC
COSN5754651	70	COSMIC
COSN22628308	350	COSMIC
COSN7045660	774	COSMIC
COSN26724821	902	COSMIC
COSN21653252	1209	COSMIC
COSN25512928	1327	COSMIC
COSN25476038	1336	COSMIC
COSN25511596	1339	COSMIC
COSN25512929	1378	COSMIC
COSN25473252	1383	COSMIC
COSN25474653	1408	COSMIC
COSN9625114	1483	COSMIC
COSN27772114	1695	COSMIC
COSN5754652	1713	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs748847688	10	dbSNP
rs1485434886	13	dbSNP
rs968030387	14	dbSNP
rs111528431	18	dbSNP
rs1392019161	26	dbSNP
rs541454355	28	dbSNP
rs1256992842	30	dbSNP
rs1482398431	34	dbSNP
rs1188570058	40	dbSNP
rs1420647180	52	dbSNP
rs190477540	56	dbSNP
rs979666432	57	dbSNP
rs926505383	64	dbSNP
rs938075860	66	dbSNP
rs931260581	68	dbSNP
rs992262027	80	dbSNP
rs917895859	82	dbSNP
rs377509849	86	dbSNP
rs1322731516	90	dbSNP
rs766284427	98	dbSNP
rs547442887	102	dbSNP
rs1002672364	105	dbSNP
rs1213545121	110	dbSNP
rs1056912940	112	dbSNP
rs894205131	113	dbSNP
rs1463124871	116	dbSNP
rs754833623	119	dbSNP
rs1188202115	124	dbSNP
rs1047971360	125	dbSNP
rs767231221	125	dbSNP
rs1259420199	126	dbSNP
rs1411368550	135	dbSNP
rs1443557731	143	dbSNP
rs570228942	154	dbSNP
rs17229438	155	dbSNP
rs1039601078	176	dbSNP
rs1266502796	181	dbSNP
rs1227348705	196	dbSNP
rs968404812	214	dbSNP
rs1157517723	227	dbSNP
rs755873342	232	dbSNP
rs574454386	237	dbSNP
rs369619235	238	dbSNP
rs117487508	251	dbSNP
rs954890107	255	dbSNP
rs2600899	271	dbSNP
rs760088462	274	dbSNP
rs2641566	278	dbSNP
rs1310341353	291	dbSNP
rs1461411240	300	dbSNP
rs2600900	301	dbSNP
rs910702328	302	dbSNP
rs140392921	308	dbSNP
rs964795316	309	dbSNP
rs765762242	318	dbSNP
rs1283696591	335	dbSNP
rs1033906735	337	dbSNP
rs554970182	343	dbSNP
rs1452768448	352	dbSNP
rs1267455033	354	dbSNP
rs1194790068	363	dbSNP
rs1338011918	373	dbSNP
rs959248356	374	dbSNP
rs1253748855	376	dbSNP
rs1347650994	383	dbSNP
rs1212152614	384	dbSNP
rs1235721785	387	dbSNP
rs753096159	393	dbSNP
rs973507209	393	dbSNP
rs1328270134	396	dbSNP
rs1368606762	400	dbSNP
rs1309796159	403	dbSNP
rs920633746	415	dbSNP
rs1394252929	418	dbSNP
rs1170385981	421	dbSNP
rs1478798395	424	dbSNP
rs568394007	436	dbSNP
rs368044220	445	dbSNP
rs1178834697	457	dbSNP
rs1180423606	472	dbSNP
rs879613181	473	dbSNP
rs1239673936	481	dbSNP
rs1481800731	484	dbSNP
rs1437460650	487	dbSNP
rs1254544476	490	dbSNP
rs1206506552	499	dbSNP
rs1485516428	500	dbSNP
rs1278620515	506	dbSNP
rs1221363096	511	dbSNP
rs929415915	514	dbSNP
rs1279238183	515	dbSNP
rs983444747	518	dbSNP
rs1472686872	528	dbSNP
rs533958841	536	dbSNP
rs1352437303	546	dbSNP
rs1290058761	549	dbSNP
rs554295198	569	dbSNP
rs1178940127	574	dbSNP
rs1404356527	575	dbSNP
rs1374835863	576	dbSNP
rs1331943297	585	dbSNP
rs1415997945	599	dbSNP
rs1402415130	609	dbSNP
rs1469644363	610	dbSNP
rs193042121	617	dbSNP
rs1413548820	620	dbSNP
rs1379328211	621	dbSNP
rs1039309116	628	dbSNP
rs546020034	636	dbSNP
rs1440325372	637	dbSNP
rs1212660888	639	dbSNP
rs938512596	640	dbSNP
rs1051919057	643	dbSNP
rs1278979496	649	dbSNP
rs1331246640	652	dbSNP
rs556440571	653	dbSNP
rs1232821091	655	dbSNP
rs1220871749	656	dbSNP
rs894297137	659	dbSNP
rs563492233	661	dbSNP
rs1340850423	676	dbSNP
rs528370836	686	dbSNP
rs1317127851	691	dbSNP
rs1198989807	696	dbSNP
rs1390472358	700	dbSNP
rs1259788032	701	dbSNP
rs201711557	709	dbSNP
rs1159224468	714	dbSNP
rs1460512996	719	dbSNP
rs1412196265	723	dbSNP
rs576313624	727	dbSNP
rs904188919	735	dbSNP
rs1246768451	742	dbSNP
rs1187130545	745	dbSNP
rs370606249	758	dbSNP
rs751687941	760	dbSNP
rs1202183589	761	dbSNP
rs1032358662	763	dbSNP
rs1480407217	767	dbSNP
rs1320033175	788	dbSNP
rs1287563831	797	dbSNP
rs1171779259	808	dbSNP
rs542061897	809	dbSNP
rs1009091204	810	dbSNP
rs1269378621	812	dbSNP
rs959429230	814	dbSNP
rs1366892389	816	dbSNP
rs1408418168	817	dbSNP
rs1017762965	818	dbSNP
rs964847606	835	dbSNP
rs973902059	837	dbSNP
rs1013423290	860	dbSNP
rs866407255	864	dbSNP
rs145504667	865	dbSNP
rs1167065073	879	dbSNP
rs1451772676	885	dbSNP
rs1350513016	892	dbSNP
rs1248824932	903	dbSNP
rs148028232	907	dbSNP
rs1248514133	908	dbSNP
rs1281482118	909	dbSNP
rs1344758311	915	dbSNP
rs983560812	931	dbSNP
rs927930124	939	dbSNP
rs772445797	949	dbSNP
rs1354444320	954	dbSNP
rs938607839	955	dbSNP
rs909183929	958	dbSNP
rs1344080118	967	dbSNP
rs1297785705	972	dbSNP
rs1233121021	979	dbSNP
rs1365117370	979	dbSNP
rs992735602	980	dbSNP
rs963416139	981	dbSNP
rs3042006	983	dbSNP
rs397836271	983	dbSNP
rs398070643	983	dbSNP
rs540703903	985	dbSNP
rs563815321	991	dbSNP
rs915786317	998	dbSNP
rs1290195114	1001	dbSNP
rs1431653648	1002	dbSNP
rs1427580626	1003	dbSNP
rs1191826056	1004	dbSNP
rs1469713922	1016	dbSNP
rs948541602	1019	dbSNP
rs1250939010	1021	dbSNP
rs141717823	1022	dbSNP
rs1219555819	1027	dbSNP
rs1267592172	1037	dbSNP
rs1483879559	1041	dbSNP
rs758626748	1042	dbSNP
rs552343282	1046	dbSNP
rs1043205030	1047	dbSNP
rs1310042186	1050	dbSNP
rs781221433	1058	dbSNP
rs1303018348	1062	dbSNP
rs377612737	1066	dbSNP
rs933606532	1068	dbSNP
rs1286939644	1070	dbSNP
rs934389113	1074	dbSNP
rs747198992	1093	dbSNP
rs1371788621	1102	dbSNP
rs1474655860	1110	dbSNP
rs913425547	1115	dbSNP
rs890112592	1119	dbSNP
rs138183864	1123	dbSNP
rs1177098064	1124	dbSNP
rs945104531	1124	dbSNP
rs374993086	1135	dbSNP
rs1017775965	1138	dbSNP
rs900677123	1140	dbSNP
rs903483213	1142	dbSNP
rs1414743184	1148	dbSNP
rs1407326797	1150	dbSNP
rs994989740	1152	dbSNP
rs1000540082	1165	dbSNP
rs1184505601	1165	dbSNP
rs368841076	1166	dbSNP
rs1301301604	1167	dbSNP
rs1365050958	1168	dbSNP
rs1400520212	1170	dbSNP
rs1300032001	1171	dbSNP
rs568672618	1172	dbSNP
rs1226483748	1173	dbSNP
rs1482164984	1179	dbSNP
rs1210404550	1182	dbSNP
rs1257353388	1182	dbSNP
rs1284586914	1187	dbSNP
rs895057313	1192	dbSNP
rs143844645	1207	dbSNP
rs1351622842	1214	dbSNP
rs74009404	1226	dbSNP
rs1024797351	1237	dbSNP
rs1381807330	1241	dbSNP
rs1290387382	1243	dbSNP
rs1488372271	1245	dbSNP
rs983925818	1246	dbSNP
rs11637137	1247	dbSNP
rs879065525	1262	dbSNP
rs879003497	1263	dbSNP
rs1161417227	1274	dbSNP
rs960693276	1275	dbSNP
rs1412559115	1278	dbSNP
rs992746869	1279	dbSNP
rs1257996354	1304	dbSNP
rs1192874192	1316	dbSNP
rs11553040	1317	dbSNP
rs11553046	1326	dbSNP
rs11553041	1333	dbSNP
rs556697892	1344	dbSNP
rs1245868083	1346	dbSNP
rs1446608464	1347	dbSNP
rs576157768	1364	dbSNP
rs535307636	1367	dbSNP
rs11553039	1368	dbSNP
rs555621791	1370	dbSNP
rs1243275607	1374	dbSNP
rs532568026	1375	dbSNP
rs1324083497	1387	dbSNP
rs34801527	1387	dbSNP
rs1420426626	1389	dbSNP
rs925766367	1391	dbSNP
rs1160201401	1404	dbSNP
rs11553047	1410	dbSNP
rs1363260509	1413	dbSNP
rs1322830953	1414	dbSNP
rs547796024	1415	dbSNP
rs1435853302	1419	dbSNP
rs1382629236	1423	dbSNP
rs1161596545	1424	dbSNP
rs11553030	1429	dbSNP
rs1456325106	1431	dbSNP
rs572277153	1438	dbSNP
rs1165191529	1447	dbSNP
rs1052800961	1449	dbSNP
rs11553036	1455	dbSNP
rs1334485339	1460	dbSNP
rs1448670783	1461	dbSNP
rs974769520	1466	dbSNP
rs1342052193	1467	dbSNP
rs7151	1473	dbSNP
rs1313981616	1477	dbSNP
rs1489734419	1479	dbSNP
rs944344954	1482	dbSNP
rs4780	1483	dbSNP
rs1340517955	1492	dbSNP
rs1258469548	1516	dbSNP
rs1349081308	1517	dbSNP
rs1205076575	1519	dbSNP
rs900727413	1522	dbSNP
rs1229427253	1526	dbSNP
rs1330809210	1532	dbSNP
rs1321637075	1534	dbSNP
rs1388649260	1537	dbSNP
rs1279768996	1551	dbSNP
rs995478153	1552	dbSNP
rs368409484	1556	dbSNP
rs767390881	1579	dbSNP
rs1428793682	1585	dbSNP
rs1368491786	1587	dbSNP
rs1170322498	1588	dbSNP
rs1475532784	1589	dbSNP
rs1242815475	1591	dbSNP
rs752591118	1592	dbSNP
rs147243617	1594	dbSNP
rs1184028670	1596	dbSNP
rs1482244480	1607	dbSNP
rs563063364	1612	dbSNP
rs1277206740	1613	dbSNP
rs1344553975	1618	dbSNP
rs960703121	1626	dbSNP
rs1273321358	1632	dbSNP
rs946221511	1637	dbSNP
rs1012224104	1639	dbSNP
rs1452892464	1649	dbSNP
rs1409124000	1650	dbSNP
rs1368698866	1657	dbSNP
rs1309582905	1659	dbSNP
rs1042496468	1666	dbSNP
rs1407993426	1668	dbSNP
rs1468085840	1678	dbSNP
rs1023622544	1679	dbSNP
rs970030519	1681	dbSNP
rs547042655	1691	dbSNP
rs1467069427	1697	dbSNP
rs936455997	1704	dbSNP
rs1392779246	1707	dbSNP
rs567911872	1725	dbSNP
rs894839858	1729	dbSNP
rs979078327	1738	dbSNP
rs1304162964	1747	dbSNP
rs1254655189	1752	dbSNP
rs1182924141	1762	dbSNP
rs879639542	1777	dbSNP
rs1054230	1778	dbSNP
rs1442239727	1793	dbSNP
rs1276688345	1794	dbSNP
rs1046363384	1795	dbSNP
rs925849515	1796	dbSNP
rs1346056114	1803	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.