ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ccUGAGAUGUCUGUGUGGa 5' ||| ||| ||||||| Target 5' gcACT-TAC-TACACACCt 3'	280 - 296	151.00	-13.10
2	miRNA 3' ccUGAGAUGUCUGUGUGGa 5' ::|: |:| :|||||: Target 5' ttGTTTCATAAGCACACTg 3'	764 - 782	121.00	-7.70
3	miRNA 3' ccugagaugucuGUGUGGa 5' |||||| Target 5' agctggagaatcCACACCg 3'	119 - 137	120.00	-13.70

Mutant ID	Mutant Position	Mutant Source
COSN30102704	19	COSMIC
COSN14210357	36	COSMIC
COSN31568753	37	COSMIC
COSN30591473	41	COSMIC
COSN31578293	46	COSMIC
COSN26981498	47	COSMIC
COSN31613239	70	COSMIC
COSN30179831	81	COSMIC
COSN31496068	126	COSMIC
COSN30143702	139	COSMIC
COSN16726941	356	COSMIC
COSN17011151	805	COSMIC
COSN22628554	1359	COSMIC
COSN9311918	1430	COSMIC
COSN5086051	1507	COSMIC
COSN31778527	1554	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1223011627	1	dbSNP
rs867005050	3	dbSNP
rs1330393333	10	dbSNP
rs765365649	13	dbSNP
rs372472253	15	dbSNP
rs186263133	18	dbSNP
rs1057194794	21	dbSNP
rs766055442	27	dbSNP
rs1419283323	31	dbSNP
rs760595549	32	dbSNP
rs544363587	36	dbSNP
rs767235308	37	dbSNP
rs761558475	41	dbSNP
rs773674531	42	dbSNP
rs768268381	46	dbSNP
rs146219124	47	dbSNP
rs1419661093	52	dbSNP
rs1158284391	56	dbSNP
rs114131556	57	dbSNP
rs1303483283	58	dbSNP
rs1472273561	60	dbSNP
rs769193146	69	dbSNP
rs1291876996	70	dbSNP
rs1375601651	77	dbSNP
rs181523053	95	dbSNP
rs572807998	96	dbSNP
rs1490571476	99	dbSNP
rs1267005266	102	dbSNP
rs1208403828	104	dbSNP
rs1352021328	108	dbSNP
rs1487074095	108	dbSNP
rs370485396	133	dbSNP
rs1039138255	134	dbSNP
rs1008577193	136	dbSNP
rs974097350	137	dbSNP
rs1234181343	147	dbSNP
rs961511130	153	dbSNP
rs1176601588	154	dbSNP
rs1016177230	156	dbSNP
rs1446330539	157	dbSNP
rs1223656039	170	dbSNP
rs1013789742	171	dbSNP
rs961169975	175	dbSNP
rs890094454	178	dbSNP
rs776572831	182	dbSNP
rs189588966	183	dbSNP
rs376016864	187	dbSNP
rs929524193	191	dbSNP
rs1356391297	196	dbSNP
rs920795527	196	dbSNP
rs1297631993	200	dbSNP
rs1375367075	202	dbSNP
rs1385422773	208	dbSNP
rs1316185669	217	dbSNP
rs541660291	228	dbSNP
rs1226296404	237	dbSNP
rs184768472	262	dbSNP
rs1408284387	264	dbSNP
rs746872571	266	dbSNP
rs1203251278	268	dbSNP
rs11418580	280	dbSNP
rs1184119673	280	dbSNP
rs1242366333	281	dbSNP
rs1178654850	290	dbSNP
rs1161726108	305	dbSNP
rs948847976	308	dbSNP
rs916012944	317	dbSNP
rs747504752	319	dbSNP
rs993300998	323	dbSNP
rs1169830994	326	dbSNP
rs960032155	327	dbSNP
rs891581591	331	dbSNP
rs538353930	332	dbSNP
rs1337931279	341	dbSNP
rs1382566583	344	dbSNP
rs1053354615	346	dbSNP
rs192755070	351	dbSNP
rs978038370	355	dbSNP
rs966615994	357	dbSNP
rs1255674150	363	dbSNP
rs1049607577	364	dbSNP
rs932581171	370	dbSNP
rs141959298	380	dbSNP
rs974086021	381	dbSNP
rs1253832065	387	dbSNP
rs1229163284	388	dbSNP
rs1193198613	401	dbSNP
rs961754882	401	dbSNP
rs537397686	403	dbSNP
rs1010725794	407	dbSNP
rs1296580800	410	dbSNP
rs960882890	410	dbSNP
rs992656337	410	dbSNP
rs964872679	411	dbSNP
rs1320711803	415	dbSNP
rs1432697573	416	dbSNP
rs1384873375	418	dbSNP
rs1429675474	420	dbSNP
rs1275875119	421	dbSNP
rs1017372665	429	dbSNP
rs188983109	431	dbSNP
rs1415065051	437	dbSNP
rs1282041994	446	dbSNP
rs576528488	448	dbSNP
rs1206761732	449	dbSNP
rs1009021572	453	dbSNP
rs1284763459	457	dbSNP
rs890125716	458	dbSNP
rs1170968168	459	dbSNP
rs115051018	464	dbSNP
rs1011275163	482	dbSNP
rs1188301323	485	dbSNP
rs993748998	491	dbSNP
rs1200239552	493	dbSNP
rs899412787	495	dbSNP
rs1038332096	501	dbSNP
rs1245465582	503	dbSNP
rs147573363	505	dbSNP
rs916024763	506	dbSNP
rs1400464298	507	dbSNP
rs536133022	519	dbSNP
rs938741397	531	dbSNP
rs1405105529	536	dbSNP
rs6906516	538	dbSNP
rs977399763	539	dbSNP
rs966644944	544	dbSNP
rs78307568	548	dbSNP
rs898453752	550	dbSNP
rs1038261176	551	dbSNP
rs1293590495	554	dbSNP
rs989779749	555	dbSNP
rs1223488637	558	dbSNP
rs1266850874	566	dbSNP
rs964576210	590	dbSNP
rs1225852321	591	dbSNP
rs1017819450	592	dbSNP
rs748567151	606	dbSNP
rs544736322	607	dbSNP
rs1315580458	608	dbSNP
rs11321300	616	dbSNP
rs532489021	623	dbSNP
rs939461728	628	dbSNP
rs1157233037	631	dbSNP
rs926760452	634	dbSNP
rs565337944	635	dbSNP
rs980976276	645	dbSNP
rs1162278735	650	dbSNP
rs1338695197	651	dbSNP
rs987213727	652	dbSNP
rs1332256361	657	dbSNP
rs1392649058	658	dbSNP
rs1328902172	660	dbSNP
rs968281084	660	dbSNP
rs1444409315	670	dbSNP
rs1022520830	673	dbSNP
rs1393262288	676	dbSNP
rs1247047131	677	dbSNP
rs954457550	683	dbSNP
rs988799838	688	dbSNP
rs1354444891	689	dbSNP
rs540269335	691	dbSNP
rs1025966659	703	dbSNP
rs955709078	705	dbSNP
rs867792847	706	dbSNP
rs184916707	718	dbSNP
rs1428164485	721	dbSNP
rs12212501	723	dbSNP
rs1016522379	724	dbSNP
rs1013551964	725	dbSNP
rs894677403	727	dbSNP
rs1472750144	728	dbSNP
rs1187222541	734	dbSNP
rs1366386933	736	dbSNP
rs1370741513	739	dbSNP
rs192785343	743	dbSNP
rs996603123	744	dbSNP
rs1057483209	746	dbSNP
rs938773962	752	dbSNP
rs1338593224	757	dbSNP
rs1383324992	759	dbSNP
rs939946390	762	dbSNP
rs887103484	763	dbSNP
rs1310259933	765	dbSNP
rs1338929237	773	dbSNP
rs903254204	778	dbSNP
rs1041794298	787	dbSNP
rs1252022913	793	dbSNP
rs1317369734	794	dbSNP
rs947387566	798	dbSNP
rs1250552725	803	dbSNP
rs1462125020	811	dbSNP
rs575145220	814	dbSNP
rs1200247054	823	dbSNP
rs914518458	833	dbSNP
rs1202609704	835	dbSNP
rs1254439574	837	dbSNP
rs570959843	839	dbSNP
rs1422348840	845	dbSNP
rs1476544453	847	dbSNP
rs750940751	847	dbSNP
rs926727878	850	dbSNP
rs1406769724	853	dbSNP
rs910424142	855	dbSNP
rs987246183	863	dbSNP
rs954518056	870	dbSNP
rs1026039188	872	dbSNP
rs1354136078	879	dbSNP
rs1281253169	883	dbSNP
rs1279040338	889	dbSNP
rs971748634	890	dbSNP
rs963086688	897	dbSNP
rs1016337425	898	dbSNP
rs868792635	906	dbSNP
rs1324901124	907	dbSNP
rs988379650	911	dbSNP
rs1254698808	914	dbSNP
rs376326712	915	dbSNP
rs1205410413	919	dbSNP
rs1408229306	929	dbSNP
rs9371017	936	dbSNP
rs1200967938	939	dbSNP
rs1429312697	941	dbSNP
rs1035889668	945	dbSNP
rs1173864734	954	dbSNP
rs9371016	956	dbSNP
rs1405184359	967	dbSNP
rs531171692	972	dbSNP
rs1412214494	973	dbSNP
rs1160825822	975	dbSNP
rs1360136702	977	dbSNP
rs986908568	978	dbSNP
rs1410595359	983	dbSNP
rs560707633	987	dbSNP
rs1481203564	988	dbSNP
rs1241362553	990	dbSNP
rs1269211601	991	dbSNP
rs1220512308	992	dbSNP
rs796897236	995	dbSNP
rs1487856506	996	dbSNP
rs1189453704	1014	dbSNP
rs764383920	1017	dbSNP
rs1252605630	1028	dbSNP
rs1027992788	1039	dbSNP
rs1286197827	1044	dbSNP
rs1418171723	1045	dbSNP
rs762601506	1062	dbSNP
rs1350586853	1066	dbSNP
rs1435873595	1073	dbSNP
rs1041825098	1074	dbSNP
rs1329860485	1077	dbSNP
rs1376177675	1079	dbSNP
rs1287796850	1080	dbSNP
rs947418778	1082	dbSNP
rs1309067023	1085	dbSNP
rs1375144693	1087	dbSNP
rs996572300	1088	dbSNP
rs1290121232	1097	dbSNP
rs962492428	1105	dbSNP
rs1347588319	1109	dbSNP
rs1218780394	1110	dbSNP
rs1017209307	1112	dbSNP
rs1480951602	1114	dbSNP
rs76493267	1122	dbSNP
rs565436543	1125	dbSNP
rs1348517819	1132	dbSNP
rs1446204207	1134	dbSNP
rs752323922	1140	dbSNP
rs1458229557	1141	dbSNP
rs910473903	1142	dbSNP
rs567255406	1149	dbSNP
rs1162791334	1150	dbSNP
rs1481153589	1154	dbSNP
rs1356345720	1157	dbSNP
rs1425382782	1162	dbSNP
rs1331228926	1165	dbSNP
rs146946922	1168	dbSNP
rs1454938771	1180	dbSNP
rs1285112989	1181	dbSNP
rs919058911	1185	dbSNP
rs971825150	1187	dbSNP
rs530412498	1190	dbSNP
rs908903187	1210	dbSNP
rs569473547	1211	dbSNP
rs991550729	1212	dbSNP
rs1197987023	1213	dbSNP
rs1256098362	1215	dbSNP
rs1462562681	1220	dbSNP
rs1210560381	1226	dbSNP
rs1464369336	1228	dbSNP
rs915397644	1233	dbSNP
rs1266208886	1236	dbSNP
rs1472627488	1238	dbSNP
rs187005958	1241	dbSNP
rs1180335739	1242	dbSNP
rs1421794737	1258	dbSNP
rs1053055690	1271	dbSNP
rs1478177887	1271	dbSNP
rs1398782707	1280	dbSNP
rs1471204803	1285	dbSNP
rs376770783	1294	dbSNP
rs1358941042	1296	dbSNP
rs1035961903	1297	dbSNP
rs1445409932	1299	dbSNP
rs1271742821	1300	dbSNP
rs1325788626	1301	dbSNP
rs1340692104	1307	dbSNP
rs1279529915	1311	dbSNP
rs565253585	1315	dbSNP
rs1003085194	1327	dbSNP
rs967544398	1334	dbSNP
rs1277741828	1335	dbSNP
rs1272270204	1339	dbSNP
rs1208346609	1340	dbSNP
rs769359074	1341	dbSNP
rs1084	1351	dbSNP
rs1193048263	1352	dbSNP
rs1011701463	1364	dbSNP
rs1421557191	1366	dbSNP
rs893191158	1370	dbSNP
rs1039734963	1373	dbSNP
rs530326680	1388	dbSNP
rs1413141397	1389	dbSNP
rs1320640991	1398	dbSNP
rs182985131	1402	dbSNP
rs1295875010	1411	dbSNP
rs920944431	1412	dbSNP
rs975120155	1422	dbSNP
rs1359248858	1424	dbSNP
rs962459566	1425	dbSNP
rs1225288903	1431	dbSNP
rs1174836671	1443	dbSNP
rs1286381866	1445	dbSNP
rs1354708194	1447	dbSNP
rs1412395736	1451	dbSNP
rs1216157437	1452	dbSNP
rs1421271079	1453	dbSNP
rs891089261	1458	dbSNP
rs1016766337	1467	dbSNP
rs1246066881	1473	dbSNP
rs1160832675	1474	dbSNP
rs560945600	1475	dbSNP
rs1051640398	1476	dbSNP
rs933230993	1478	dbSNP
rs1418785785	1480	dbSNP
rs1156430246	1483	dbSNP
rs951254129	1497	dbSNP
rs1484240004	1500	dbSNP
rs1238125010	1503	dbSNP
rs1035308051	1507	dbSNP
rs919132808	1508	dbSNP
rs1309796420	1509	dbSNP
rs1375836880	1510	dbSNP
rs1310638210	1512	dbSNP
rs1036138083	1519	dbSNP
rs1284172837	1526	dbSNP
rs369165731	1537	dbSNP
rs908966735	1541	dbSNP
rs542555402	1542	dbSNP
rs1229543733	1546	dbSNP
rs776544318	1549	dbSNP
rs958616221	1552	dbSNP
rs770911508	1556	dbSNP
rs1181051425	1558	dbSNP
rs981724609	1564	dbSNP
rs1455057762	1570	dbSNP
rs1045117203	1572	dbSNP
rs1177111444	1572	dbSNP
rs145428957	1572	dbSNP
rs369635810	1572	dbSNP
rs575460322	1572	dbSNP
rs772191660	1572	dbSNP
rs760618976	1580	dbSNP
rs1301728876	1586	dbSNP
rs1399392434	1591	dbSNP
rs1444343656	1597	dbSNP
rs541598091	1603	dbSNP
rs141233146	1604	dbSNP
rs2038744	1621	dbSNP
rs1428804894	1637	dbSNP
rs577733844	1640	dbSNP
rs891160606	1644	dbSNP
rs1259296314	1645	dbSNP
rs1457682116	1648	dbSNP
rs756949685	1671	dbSNP
rs1051046209	1687	dbSNP
rs997414747	1693	dbSNP
rs145798863	1697	dbSNP
rs534685244	1698	dbSNP
rs1186359722	1706	dbSNP
rs567316638	1708	dbSNP
rs897735836	1711	dbSNP
rs555390198	1727	dbSNP

- 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.

- 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.

- 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.