ID	Duplex structure	Position	Score	MFE
1	miRNA 3' agaugagguuuuccGAUGUUAGu 5' :|:||||| Target 5' tcatttggtcatccTTGCAATCc 3'	1123 - 1145	125.00	-7.50
2	miRNA 3' agaUGA-GGUUUUCCG------AUGUUAGu 5' ||| : ||||| | |||||| Target 5' tcaACTGTGAAAAGACACATCACACAATCa 3'	310 - 339	122.00	-9.40
3	miRNA 3' agAUGAGGUUUUCCGAUG-UUAGu 5' |||| : ||| | ||| |||: Target 5' gaTACTGT-AAATGATACTAATTt 3'	148 - 170	116.00	-6.60

Mutant ID	Mutant Position	Mutant Source
COSN24307096	3	COSMIC
COSN26998937	22	COSMIC
COSN30476187	28	COSMIC
COSN30505513	31	COSMIC
COSN30458627	32	COSMIC
COSN30458620	33	COSMIC
COSN30169210	52	COSMIC
COSN31567363	75	COSMIC
COSN13867558	88	COSMIC
COSN26998936	89	COSMIC
COSN30144721	107	COSMIC
COSN30502742	112	COSMIC
COSN30184289	115	COSMIC
COSN31559335	244	COSMIC
COSN28824255	287	COSMIC
COSN26563719	360	COSMIC
COSN28677500	382	COSMIC
COSN26325394	427	COSMIC
COSN22596479	456	COSMIC
COSN31579035	497	COSMIC
COSN31519043	505	COSMIC
COSN31512891	513	COSMIC
COSN28648771	522	COSMIC
COSN8000421	570	COSMIC
COSN26665932	737	COSMIC
COSN26665931	752	COSMIC
COSN21694650	755	COSMIC
COSN26646230	761	COSMIC
COSN31608558	764	COSMIC
COSN26665930	788	COSMIC
COSN9141735	827	COSMIC
COSN26644422	881	COSMIC
COSN26637891	882	COSMIC
COSN31585404	906	COSMIC
COSN26580109	945	COSMIC
COSN31484609	1058	COSMIC
COSN8899531	1144	COSMIC
COSN30176705	1286	COSMIC
COSN28402157	1322	COSMIC
COSN26548224	1348	COSMIC
COSN31596053	1392	COSMIC
COSN31529740	1404	COSMIC
COSN19660945	1406	COSMIC
COSN31518694	1441	COSMIC
COSN31529170	1498	COSMIC
COSN23222315	1617	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1327160254	7	dbSNP
rs750111448	8	dbSNP
rs765043055	13	dbSNP
rs761226884	15	dbSNP
rs1472196388	16	dbSNP
rs1363988460	18	dbSNP
rs1183602229	20	dbSNP
rs200602780	21	dbSNP
rs528812995	23	dbSNP
rs1214104193	26	dbSNP
rs1469722543	27	dbSNP
rs760688909	28	dbSNP
rs201295239	29	dbSNP
rs187204768	32	dbSNP
rs1262699317	33	dbSNP
rs773415961	35	dbSNP
rs1194254182	36	dbSNP
rs779631764	36	dbSNP
rs1376444799	38	dbSNP
rs371693730	39	dbSNP
rs899700274	40	dbSNP
rs748150641	42	dbSNP
rs368141343	43	dbSNP
rs1005431935	56	dbSNP
rs780048962	58	dbSNP
rs1351757693	61	dbSNP
rs182869336	71	dbSNP
rs557493991	88	dbSNP
rs1369437294	89	dbSNP
rs1242899650	93	dbSNP
rs73611765	104	dbSNP
rs73611764	110	dbSNP
rs1366614450	111	dbSNP
rs1270503795	112	dbSNP
rs1432674901	115	dbSNP
rs1343596496	121	dbSNP
rs1049065708	124	dbSNP
rs1448546963	130	dbSNP
rs929329105	135	dbSNP
rs897810106	139	dbSNP
rs1056989975	144	dbSNP
rs527244006	152	dbSNP
rs939444041	153	dbSNP
rs1416251387	170	dbSNP
rs544387060	172	dbSNP
rs1377397162	175	dbSNP
rs577095993	180	dbSNP
rs1172293414	189	dbSNP
rs148757188	198	dbSNP
rs191054982	208	dbSNP
rs1449714514	210	dbSNP
rs1330103976	214	dbSNP
rs41283596	215	dbSNP
rs1474636788	229	dbSNP
rs73611763	237	dbSNP
rs946969330	238	dbSNP
rs915467088	253	dbSNP
rs1272049911	261	dbSNP
rs1331994721	265	dbSNP
rs1200527091	266	dbSNP
rs988379194	268	dbSNP
rs1437104695	280	dbSNP
rs1179400646	283	dbSNP
rs554507531	288	dbSNP
rs1029326485	303	dbSNP
rs976940985	314	dbSNP
rs1467243302	315	dbSNP
rs1169053770	319	dbSNP
rs1396425563	338	dbSNP
rs1485693876	341	dbSNP
rs1408641796	355	dbSNP
rs963794287	357	dbSNP
rs1356483318	358	dbSNP
rs1018084315	361	dbSNP
rs1292292850	365	dbSNP
rs1353088599	367	dbSNP
rs1219950926	368	dbSNP
rs1251317536	378	dbSNP
rs8585	382	dbSNP
rs1193955471	383	dbSNP
rs775806445	384	dbSNP
rs1485858324	389	dbSNP
rs1025541554	392	dbSNP
rs1255651312	396	dbSNP
rs1478890544	396	dbSNP
rs1216093844	401	dbSNP
rs1372533106	406	dbSNP
rs144341721	413	dbSNP
rs1447090241	418	dbSNP
rs1277771319	420	dbSNP
rs1174801340	426	dbSNP
rs1376519226	430	dbSNP
rs897855587	443	dbSNP
rs1311381438	445	dbSNP
rs73611762	458	dbSNP
rs149631469	466	dbSNP
rs1383519690	467	dbSNP
rs879941567	478	dbSNP
rs1311098113	479	dbSNP
rs1225201538	482	dbSNP
rs1056436022	483	dbSNP
rs1305530392	490	dbSNP
rs1348394533	494	dbSNP
rs1209902067	498	dbSNP
rs1465608196	505	dbSNP
rs747116362	506	dbSNP
rs1205887038	512	dbSNP
rs905254358	513	dbSNP
rs1426811509	514	dbSNP
rs1045270254	517	dbSNP
rs1049679	522	dbSNP
rs1454169890	523	dbSNP
rs915393538	532	dbSNP
rs1053153792	533	dbSNP
rs73611761	536	dbSNP
rs1455069553	538	dbSNP
rs935587429	545	dbSNP
rs759598047	548	dbSNP
rs1383697942	549	dbSNP
rs922267379	550	dbSNP
rs73611760	552	dbSNP
rs1327261702	567	dbSNP
rs976404142	568	dbSNP
rs964150271	570	dbSNP
rs73611759	571	dbSNP
rs1259221627	582	dbSNP
rs1185068827	584	dbSNP
rs774173278	585	dbSNP
rs1260353908	592	dbSNP
rs1482676227	595	dbSNP
rs1211720825	599	dbSNP
rs1233380662	613	dbSNP
rs1253661334	619	dbSNP
rs73611758	622	dbSNP
rs1468447440	624	dbSNP
rs1192284516	632	dbSNP
rs1240455774	633	dbSNP
rs73611757	640	dbSNP
rs1305526283	641	dbSNP
rs752721921	651	dbSNP
rs73611756	655	dbSNP
rs1166502575	662	dbSNP
rs1394510605	664	dbSNP
rs1440138786	665	dbSNP
rs868658306	674	dbSNP
rs6095755	676	dbSNP
rs1375335838	677	dbSNP
rs1391777014	678	dbSNP
rs1025425012	680	dbSNP
rs1235316253	681	dbSNP
rs1337135154	683	dbSNP
rs994499953	689	dbSNP
rs1212438777	690	dbSNP
rs959945210	694	dbSNP
rs1481212921	695	dbSNP
rs80158980	707	dbSNP
rs1176991228	716	dbSNP
rs1374234484	717	dbSNP
rs1189594783	720	dbSNP
rs770370283	720	dbSNP
rs1003516625	725	dbSNP
rs1440716704	726	dbSNP
rs528773295	733	dbSNP
rs73611755	737	dbSNP
rs754594279	740	dbSNP
rs114097793	742	dbSNP
rs894010518	746	dbSNP
rs1162340301	755	dbSNP
rs1370882745	759	dbSNP
rs73611754	761	dbSNP
rs181019888	762	dbSNP
rs1294129970	768	dbSNP
rs1408137007	770	dbSNP
rs59124899	782	dbSNP
rs73611753	784	dbSNP
rs1231543169	786	dbSNP
rs1275125492	791	dbSNP
rs1308627808	793	dbSNP
rs1204375556	796	dbSNP
rs1250024552	804	dbSNP
rs1456351686	814	dbSNP
rs1237495207	815	dbSNP
rs1486838574	816	dbSNP
rs1214022611	818	dbSNP
rs1257803242	820	dbSNP
rs935583318	830	dbSNP
rs1271508513	837	dbSNP
rs1244392235	841	dbSNP
rs1317572762	849	dbSNP
rs374126556	852	dbSNP
rs545495098	853	dbSNP
rs1169620042	856	dbSNP
rs1352615743	859	dbSNP
rs1436338365	859	dbSNP
rs1313200964	861	dbSNP
rs1375075469	863	dbSNP
rs751129605	864	dbSNP
rs1413119490	874	dbSNP
rs1272020927	875	dbSNP
rs1041683241	878	dbSNP
rs578177787	886	dbSNP
rs1376727045	888	dbSNP
rs777590807	892	dbSNP
rs558706247	895	dbSNP
rs942395810	897	dbSNP
rs1280016716	904	dbSNP
rs1392791136	909	dbSNP
rs1361971820	912	dbSNP
rs1243387885	918	dbSNP
rs1485907657	920	dbSNP
rs1185916313	925	dbSNP
rs1388262194	926	dbSNP
rs1455480410	928	dbSNP
rs1174063318	940	dbSNP
rs1394193833	946	dbSNP
rs910887394	954	dbSNP
rs1320495288	955	dbSNP
rs1345930033	957	dbSNP
rs1430124432	962	dbSNP
rs1287125744	968	dbSNP
rs983869694	971	dbSNP
rs1291407801	973	dbSNP
rs1303193371	981	dbSNP
rs1346130710	983	dbSNP
rs1457052224	986	dbSNP
rs952432084	989	dbSNP
rs73611752	990	dbSNP
rs755701683	996	dbSNP
rs1317077628	1012	dbSNP
rs879458095	1019	dbSNP
rs1469784005	1021	dbSNP
rs1437288446	1022	dbSNP
rs540060498	1023	dbSNP
rs1160571546	1025	dbSNP
rs573044579	1033	dbSNP
rs1408994300	1041	dbSNP
rs1400303487	1044	dbSNP
rs1157068231	1047	dbSNP
rs1396766423	1056	dbSNP
rs1442334469	1063	dbSNP
rs1325821177	1068	dbSNP
rs1369319531	1073	dbSNP
rs190672140	1074	dbSNP
rs1444828879	1076	dbSNP
rs1280434152	1082	dbSNP
rs1373645674	1091	dbSNP
rs972570791	1099	dbSNP
rs1194262082	1117	dbSNP
rs765923024	1130	dbSNP
rs1265745096	1133	dbSNP
rs1455399165	1136	dbSNP
rs762699970	1141	dbSNP
rs1265068992	1146	dbSNP
rs1209049303	1147	dbSNP
rs535813598	1151	dbSNP
rs1233198266	1158	dbSNP
rs960241899	1164	dbSNP
rs1180249701	1188	dbSNP
rs568498428	1197	dbSNP
rs1486294844	1198	dbSNP
rs1164232469	1203	dbSNP
rs556570713	1205	dbSNP
rs1412091779	1207	dbSNP
rs1262719681	1210	dbSNP
rs1329996253	1217	dbSNP
rs980407866	1220	dbSNP
rs1213660633	1225	dbSNP
rs1353150640	1227	dbSNP
rs2733	1233	dbSNP
rs1233380045	1238	dbSNP
rs1292094517	1241	dbSNP
rs535738819	1244	dbSNP
rs1369457027	1245	dbSNP
rs1011001131	1265	dbSNP
rs765686922	1266	dbSNP
rs1031603733	1267	dbSNP
rs1203354214	1273	dbSNP
rs15218	1282	dbSNP
rs115164526	1285	dbSNP
rs1290320039	1286	dbSNP
rs1475619019	1289	dbSNP
rs901321066	1293	dbSNP
rs73611751	1295	dbSNP
rs1476701953	1299	dbSNP
rs1168962884	1306	dbSNP
rs368384878	1309	dbSNP
rs186621934	1313	dbSNP
rs1376935527	1315	dbSNP
rs762056905	1317	dbSNP
rs1436456382	1320	dbSNP
rs1332988963	1321	dbSNP
rs777176288	1322	dbSNP
rs942949266	1323	dbSNP
rs1270685435	1324	dbSNP
rs1363373433	1335	dbSNP
rs374324590	1337	dbSNP
rs1428912944	1341	dbSNP
rs1225910254	1354	dbSNP
rs1282143095	1355	dbSNP
rs1486133858	1362	dbSNP
rs1210412389	1366	dbSNP
rs1254217844	1375	dbSNP
rs889457438	1375	dbSNP
rs1191826713	1381	dbSNP
rs1487990461	1385	dbSNP
rs1373022045	1398	dbSNP
rs1448129799	1401	dbSNP
rs1281066370	1405	dbSNP
rs1213149742	1406	dbSNP
rs35338409	1406	dbSNP
rs1343402798	1408	dbSNP
rs1255931478	1409	dbSNP
rs1399513469	1410	dbSNP
rs1048011735	1411	dbSNP
rs931083930	1412	dbSNP
rs201099481	1421	dbSNP
rs1271986371	1422	dbSNP
rs1353042650	1422	dbSNP
rs1432746752	1422	dbSNP
rs566510389	1422	dbSNP
rs74435910	1422	dbSNP
rs878876092	1422	dbSNP
rs1328625645	1423	dbSNP
rs1205794427	1424	dbSNP
rs972879830	1424	dbSNP
rs1297242339	1427	dbSNP
rs752330982	1427	dbSNP
rs1397809291	1428	dbSNP
rs1339472928	1430	dbSNP
rs1174333184	1434	dbSNP
rs1312916426	1434	dbSNP
rs1375579599	1442	dbSNP
rs2664563	1444	dbSNP
rs1454988695	1452	dbSNP
rs938551200	1460	dbSNP
rs1387243547	1463	dbSNP
rs1455608963	1484	dbSNP
rs1327830454	1490	dbSNP
rs1371532175	1495	dbSNP
rs1386020825	1504	dbSNP
rs1298832061	1509	dbSNP
rs1240003244	1512	dbSNP
rs1337784130	1512	dbSNP
rs1049871	1514	dbSNP
rs1327157604	1518	dbSNP
rs1216160245	1521	dbSNP
rs2664532	1524	dbSNP
rs1274370806	1536	dbSNP
rs1208925504	1542	dbSNP
rs1189876483	1543	dbSNP
rs1253044829	1555	dbSNP
rs1420551660	1557	dbSNP
rs1171986317	1558	dbSNP
rs1182039828	1561	dbSNP
rs1462301497	1567	dbSNP
rs980050190	1569	dbSNP
rs1395129720	1571	dbSNP
rs1457306679	1576	dbSNP
rs1291620766	1579	dbSNP
rs1398328634	1584	dbSNP
rs761101085	1590	dbSNP
rs969926367	1594	dbSNP
rs1374631996	1600	dbSNP
rs1168298684	1612	dbSNP
rs567922169	1613	dbSNP
rs1258489626	1614	dbSNP
rs989456149	1616	dbSNP
rs1190084659	1617	dbSNP
rs1235224359	1618	dbSNP
rs1293945527	1626	dbSNP
rs10567504	1627	dbSNP
rs1219973741	1627	dbSNP
rs1253488306	1627	dbSNP
rs1322759081	1629	dbSNP
rs1481160284	1630	dbSNP
rs1205497350	1632	dbSNP
rs201015788	1632	dbSNP
rs1044975480	1633	dbSNP
rs1189482605	1637	dbSNP
rs775575773	1639	dbSNP
rs754458946	1645	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 research, 2016

DNA damage activates TP53-regulated surveillance mechanisms that are crucial in suppressing tumorigenesis. TP53 orchestrates these responses directly by transcriptionally modulating genes, including microRNAs (miRNAs), and by regulating miRNA biogenesis through interacting with the DROSHA complex. However, whether the association between miRNAs and AGO2 is regulated following DNA damage is not yet known. Here, we show that, following DNA damage, TP53 interacts with AGO2 to induce or reduce AGO2's association of a subset of miRNAs, including multiple let-7 family members. Furthermore, we show that specific mutations in TP53 decrease rather than increase the association of let-7 family miRNAs, reducing their activity without preventing TP53 from interacting with AGO2. This is consistent with the oncogenic properties of these mutants. Using AGO2 RIP-seq and PAR-CLIP-seq, we show that the DNA damage-induced increase in binding of let-7 family members to the RISC complex is functional. We unambiguously determine the global miRNA-mRNA interaction networks involved in the DNA damage response, validating them through the identification of miRNA-target chimeras formed by endogenous ligation reactions. We find that the target complementary region of the let-7 seed tends to have highly fixed positions and more variable ones. Additionally, we observe that miRNAs, whose cellular abundance or differential association with AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits. TP53-mediated regulation of AGO2-miRNA interaction represents a new mechanism of miRNA regulation in carcinogenesis.

- Neoplasia (New York, N.Y.), 2016

MicroRNA (miRNA) deregulation in prostate cancer (PCa) contributes to PCa initiation and metastatic progression. To comprehensively define the cancer-associated changes in miRNA targeting and function in commonly studied models of PCa, we performed photoactivatable ribonucleoside-enhanced cross-linking immunoprecipitation of the Argonaute protein in a panel of PCa cell lines modeling different stages of PCa progression. Using this comprehensive catalogue of miRNA targets, we analyzed miRNA targeting on known drivers of PCa and examined tissue-specific and stage-specific pathway targeting by miRNAs. We found that androgen receptor is the most frequently targeted PCa oncogene and that miR-148a targets the largest number of known PCa drivers. Globally, tissue-specific and stage-specific changes in miRNA targeting are driven by homeostatic response to active oncogenic pathways. Our findings indicate that, even in advanced PCa, the miRNA pool adapts to regulate continuing alterations in the cancer genome to balance oncogenic molecular changes. These findings are important because they are the first to globally characterize miRNA changes in PCa and demonstrate how the miRNA target spectrum responds to staged tumorigenesis.