ID	Duplex structure	Position	Score	MFE
1	miRNA 3' auCGAGGAAAGGUGUCUGUCCa 5' |: ||| |||| ||||||| Target 5' gaGTACCTGTCCA-AGACAGGg 3'	468 - 488	167.00	-21.50
2	miRNA 3' aucGAGGAA-AGGU------GUCUGUCca 5' :||||| |:|| :|||||| Target 5' tctTTCCTTGTTCAGCAGGCTAGACAGaa 3'	556 - 584	137.00	-14.37
3	miRNA 3' aucgagGAAAGGU------G---UCUGUCCa 5' ||||||| | ||||:|| Target 5' tgttagCTTTCCAAGGAAGCAGAAGACGGGa 3'	11 - 41	134.00	-18.60

Mutant ID	Mutant Position	Mutant Source
COSN31509545	2	COSMIC
COSN30532162	6	COSMIC
COSN26967884	34	COSMIC
COSN30185293	59	COSMIC
COSN30156978	80	COSMIC
COSN30508127	94	COSMIC
COSN31586821	95	COSMIC
COSN31538035	119	COSMIC
COSN1248326	135	COSMIC
COSN31538661	151	COSMIC
COSN507129	171	COSMIC
COSN28846379	172	COSMIC
COSN30578303	180	COSMIC
COSN14533848	302	COSMIC
COSN31528635	439	COSMIC
COSN31564971	509	COSMIC
COSN15661665	527	COSMIC
COSN29467785	534	COSMIC
COSN20079721	543	COSMIC
COSN5969778	803	COSMIC
COSN5764517	866	COSMIC
COSN17183422	871	COSMIC
COSN20116433	917	COSMIC
COSN27379549	918	COSMIC
COSN31588984	1025	COSMIC
COSN8610897	1245	COSMIC
COSN26668722	1419	COSMIC
COSN26650405	1426	COSMIC
COSN30159679	1445	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs753984398	1	dbSNP
rs777704044	2	dbSNP
rs894761562	9	dbSNP
rs756036540	10	dbSNP
rs1175981051	12	dbSNP
rs752488127	13	dbSNP
rs1410008006	16	dbSNP
rs767266911	24	dbSNP
rs759159558	25	dbSNP
rs1454496676	32	dbSNP
rs369506114	37	dbSNP
rs766032005	38	dbSNP
rs1055783035	40	dbSNP
rs1410264706	44	dbSNP
rs762381547	45	dbSNP
rs1165753619	50	dbSNP
rs772628077	51	dbSNP
rs200769415	56	dbSNP
rs1352386432	62	dbSNP
rs1438090782	63	dbSNP
rs560027205	71	dbSNP
rs930065687	85	dbSNP
rs547832793	88	dbSNP
rs767604296	92	dbSNP
rs976360147	93	dbSNP
rs1461255187	95	dbSNP
rs184352665	98	dbSNP
rs1272307686	102	dbSNP
rs1436157334	105	dbSNP
rs1223999891	111	dbSNP
rs944620654	113	dbSNP
rs1490282048	115	dbSNP
rs1197762720	116	dbSNP
rs1047802013	117	dbSNP
rs910488496	118	dbSNP
rs750843704	119	dbSNP
rs1167854316	120	dbSNP
rs1421676633	121	dbSNP
rs1302850723	123	dbSNP
rs541258422	123	dbSNP
rs532804714	124	dbSNP
rs565483019	125	dbSNP
rs545876589	126	dbSNP
rs1434492680	127	dbSNP
rs1351847409	130	dbSNP
rs1402870456	135	dbSNP
rs1299871516	136	dbSNP
rs199557267	139	dbSNP
rs986056567	142	dbSNP
rs910992105	145	dbSNP
rs767666306	149	dbSNP
rs1268376271	151	dbSNP
rs193121089	166	dbSNP
rs1230147233	167	dbSNP
rs1292736065	172	dbSNP
rs372362652	172	dbSNP
rs977134447	175	dbSNP
rs1369138652	176	dbSNP
rs1171007890	182	dbSNP
rs1220699889	184	dbSNP
rs967155351	186	dbSNP
rs1447941124	191	dbSNP
rs1010579715	200	dbSNP
rs959958043	207	dbSNP
rs776300173	208	dbSNP
rs1240884888	214	dbSNP
rs1473477596	220	dbSNP
rs1035037055	227	dbSNP
rs1191221454	250	dbSNP
rs1422303767	252	dbSNP
rs1024476304	254	dbSNP
rs541837470	266	dbSNP
rs1381462463	268	dbSNP
rs905182447	270	dbSNP
rs147743904	271	dbSNP
rs1203431899	282	dbSNP
rs1454642636	283	dbSNP
rs1005631027	291	dbSNP
rs145689733	301	dbSNP
rs41304413	304	dbSNP
rs930231392	305	dbSNP
rs888410434	318	dbSNP
rs1282770062	324	dbSNP
rs1041262029	327	dbSNP
rs1347061803	334	dbSNP
rs576587881	343	dbSNP
rs1279620963	347	dbSNP
rs1184769698	349	dbSNP
rs147934086	350	dbSNP
rs930059009	351	dbSNP
rs532329103	357	dbSNP
rs1241914915	370	dbSNP
rs901276856	379	dbSNP
rs1180072757	386	dbSNP
rs1040195226	389	dbSNP
rs978922819	396	dbSNP
rs1452745597	419	dbSNP
rs537116871	426	dbSNP
rs569841877	427	dbSNP
rs944506699	437	dbSNP
rs1156665198	442	dbSNP
rs910402874	446	dbSNP
rs986005282	449	dbSNP
rs935909534	462	dbSNP
rs770963395	463	dbSNP
rs925603771	464	dbSNP
rs1382071135	492	dbSNP
rs1441814489	500	dbSNP
rs548270614	509	dbSNP
rs977126479	512	dbSNP
rs967138515	521	dbSNP
rs1349002878	528	dbSNP
rs372613463	529	dbSNP
rs1310309729	530	dbSNP
rs536687636	533	dbSNP
rs1001445999	534	dbSNP
rs1459575354	539	dbSNP
rs1200026755	542	dbSNP
rs1250790351	544	dbSNP
rs1418204476	554	dbSNP
rs3795147	567	dbSNP
rs959103512	574	dbSNP
rs548025520	576	dbSNP
rs1375396320	586	dbSNP
rs1173294996	589	dbSNP
rs1480657002	596	dbSNP
rs1175552805	598	dbSNP
rs1023600985	617	dbSNP
rs771808212	618	dbSNP
rs1300034093	622	dbSNP
rs188916233	627	dbSNP
rs1390974980	633	dbSNP
rs1425324937	642	dbSNP
rs1034484416	649	dbSNP
rs1176258122	652	dbSNP
rs1224562464	657	dbSNP
rs1006058044	659	dbSNP
rs41308719	671	dbSNP
rs1322116582	677	dbSNP
rs1468344275	678	dbSNP
rs149685011	681	dbSNP
rs1182642406	682	dbSNP
rs1193673724	684	dbSNP
rs953003892	692	dbSNP
rs1269045543	695	dbSNP
rs1026248298	703	dbSNP
rs550420015	704	dbSNP
rs1278139107	707	dbSNP
rs747821641	712	dbSNP
rs1427102717	714	dbSNP
rs530601771	715	dbSNP
rs77057688	719	dbSNP
rs942918320	729	dbSNP
rs1461567248	730	dbSNP
rs11557678	731	dbSNP
rs139517024	736	dbSNP
rs1405928134	739	dbSNP
rs1302986278	742	dbSNP
rs574327416	743	dbSNP
rs1223426464	745	dbSNP
rs1006976333	748	dbSNP
rs1296955565	754	dbSNP
rs754411594	762	dbSNP
rs1474173630	763	dbSNP
rs1042702234	764	dbSNP
rs1221303021	778	dbSNP
rs7351369	779	dbSNP
rs947145533	780	dbSNP
rs748895538	783	dbSNP
rs1186986178	786	dbSNP
rs1261017547	795	dbSNP
rs1301586704	798	dbSNP
rs1050282385	801	dbSNP
rs780410669	802	dbSNP
rs1359236553	804	dbSNP
rs1168509313	807	dbSNP
rs34284533	808	dbSNP
rs1387298307	810	dbSNP
rs935902694	815	dbSNP
rs1455262245	825	dbSNP
rs1455691744	832	dbSNP
rs1289936647	833	dbSNP
rs1361914538	834	dbSNP
rs1456766581	835	dbSNP
rs925885351	848	dbSNP
rs545888595	849	dbSNP
rs988953623	852	dbSNP
rs111568189	856	dbSNP
rs1802291	862	dbSNP
rs543008260	866	dbSNP
rs1474738630	867	dbSNP
rs928451586	871	dbSNP
rs150498824	872	dbSNP
rs371280661	875	dbSNP
rs1483575535	877	dbSNP
rs576747236	878	dbSNP
rs916939738	881	dbSNP
rs1244974105	887	dbSNP
rs992948870	889	dbSNP
rs1238057835	890	dbSNP
rs1215185187	891	dbSNP
rs1463681184	895	dbSNP
rs1270218181	899	dbSNP
rs1229298784	910	dbSNP
rs969928088	912	dbSNP
rs558307625	913	dbSNP
rs1334601869	918	dbSNP
rs386393715	919	dbSNP
rs1160371257	920	dbSNP
rs1344990859	921	dbSNP
rs1311114767	923	dbSNP
rs386393714	923	dbSNP
rs11474161	928	dbSNP
rs958490722	931	dbSNP
rs11473736	932	dbSNP
rs1312422731	933	dbSNP
rs1491338601	933	dbSNP
rs398071578	933	dbSNP
rs74179870	933	dbSNP
rs769990752	933	dbSNP
rs770501056	933	dbSNP
rs927463028	936	dbSNP
rs1375160646	940	dbSNP
rs1224119165	941	dbSNP
rs536877098	942	dbSNP
rs576116651	945	dbSNP
rs1025822848	949	dbSNP
rs994445512	950	dbSNP
rs952801650	952	dbSNP
rs7508803	961	dbSNP
rs965717054	964	dbSNP
rs1019618156	969	dbSNP
rs1390251030	972	dbSNP
rs1007376161	975	dbSNP
rs1178004765	976	dbSNP
rs1407668234	987	dbSNP
rs901354250	997	dbSNP
rs767893431	999	dbSNP
rs555095158	1004	dbSNP
rs142509375	1007	dbSNP
rs1460648145	1010	dbSNP
rs1019708004	1015	dbSNP
rs1366491847	1020	dbSNP
rs1166655633	1023	dbSNP
rs757669747	1025	dbSNP
rs1424274941	1026	dbSNP
rs1050234503	1031	dbSNP
rs1370843992	1036	dbSNP
rs1418752830	1045	dbSNP
rs777182072	1049	dbSNP
rs1322814788	1051	dbSNP
rs751750161	1051	dbSNP
rs904372917	1052	dbSNP
rs1222963659	1060	dbSNP
rs1276514093	1065	dbSNP
rs770983647	1066	dbSNP
rs1205431765	1069	dbSNP
rs566025627	1076	dbSNP
rs575979488	1080	dbSNP
rs1482260853	1082	dbSNP
rs916868662	1093	dbSNP
rs1430594685	1103	dbSNP
rs758326545	1103	dbSNP
rs890015847	1106	dbSNP
rs1056835079	1118	dbSNP
rs1171340508	1119	dbSNP
rs547492466	1126	dbSNP
rs539312771	1127	dbSNP
rs563670415	1131	dbSNP
rs927073493	1133	dbSNP
rs1169284076	1134	dbSNP
rs891449324	1140	dbSNP
rs983848327	1141	dbSNP
rs1406534393	1142	dbSNP
rs931458558	1142	dbSNP
rs1344648972	1149	dbSNP
rs572000047	1150	dbSNP
rs973358832	1153	dbSNP
rs1271882835	1155	dbSNP
rs1341538462	1160	dbSNP
rs1802289	1165	dbSNP
rs1020029632	1173	dbSNP
rs1322802745	1178	dbSNP
rs928459910	1183	dbSNP
rs762933612	1198	dbSNP
rs954099040	1201	dbSNP
rs1297776420	1204	dbSNP
rs139124288	1205	dbSNP
rs1475436251	1207	dbSNP
rs1185493411	1211	dbSNP
rs1387919394	1217	dbSNP
rs561325863	1218	dbSNP
rs548226998	1219	dbSNP
rs1020125624	1221	dbSNP
rs878930648	1228	dbSNP
rs972801331	1232	dbSNP
rs1010563224	1243	dbSNP
rs1327022120	1244	dbSNP
rs965406906	1247	dbSNP
rs1019761705	1252	dbSNP
rs1363124121	1253	dbSNP
rs1248395527	1254	dbSNP
rs895773778	1258	dbSNP
rs760728407	1259	dbSNP
rs936993023	1265	dbSNP
rs529766737	1267	dbSNP
rs1353758949	1268	dbSNP
rs1286945342	1269	dbSNP
rs184479311	1271	dbSNP
rs1166628464	1274	dbSNP
rs1451329768	1284	dbSNP
rs1392033275	1295	dbSNP
rs1245591824	1305	dbSNP
rs905554645	1307	dbSNP
rs1189314255	1312	dbSNP
rs117470892	1315	dbSNP
rs3752311	1317	dbSNP
rs564874062	1330	dbSNP
rs1385420507	1332	dbSNP
rs201112049	1334	dbSNP
rs376330380	1339	dbSNP
rs944270620	1339	dbSNP
rs192632385	1340	dbSNP
rs912843500	1341	dbSNP
rs1340163098	1346	dbSNP
rs1488997355	1359	dbSNP
rs1021912759	1360	dbSNP
rs1351852263	1363	dbSNP
rs1274442495	1366	dbSNP
rs1205011883	1370	dbSNP
rs764720048	1386	dbSNP
rs1203626565	1392	dbSNP
rs1462247930	1396	dbSNP
rs986131451	1400	dbSNP
rs1203832349	1404	dbSNP
rs1274656519	1406	dbSNP
rs1440284003	1408	dbSNP
rs1232968816	1410	dbSNP
rs1364121764	1410	dbSNP
rs954006185	1411	dbSNP
rs1156829348	1428	dbSNP
rs1032349424	1435	dbSNP
rs1431406836	1442	dbSNP
rs1470994390	1444	dbSNP
rs1012240750	1448	dbSNP
rs1302929002	1449	dbSNP
rs141868791	1450	dbSNP
rs1043373	1455	dbSNP
rs1053165194	1456	dbSNP
rs1336706539	1468	dbSNP
rs1228210533	1474	dbSNP
rs1287002541	1490	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.