ID	Duplex structure	Position	Score	MFE
1	miRNA 3' acAUGUCGUCCGUGUCUGUCc 5' |||| || ||||| || Target 5' tgTACA--AGTAACAGAGAGg 3'	565 - 583	127.00	-17.00
2	miRNA 3' acAUGUCGUCCGUGUCUGUCc 5' |:| :: ||||:|| || Target 5' ctTGCTCTGAGCACGGAGAGc 3'	754 - 774	111.00	-12.30
3	miRNA 3' acaugucguccgUGUCUGUCc 5' :||||| | Target 5' gatccctccaaaGCAGACCGg 3'	1049 - 1069	109.00	-9.50

Mutant ID	Mutant Position	Mutant Source
COSN20490249	23	COSMIC
COSN20351046	32	COSMIC
COSN31482367	48	COSMIC
COSN31596653	78	COSMIC
COSN22216313	121	COSMIC
COSN30136014	133	COSMIC
COSN22843478	141	COSMIC
COSN1364456	156	COSMIC
COSN9261970	617	COSMIC
COSN9633931	633	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs376953907	4	dbSNP
rs1409309777	5	dbSNP
rs371595233	20	dbSNP
rs1440103828	22	dbSNP
rs1323714671	23	dbSNP
rs748436262	24	dbSNP
rs770009701	25	dbSNP
rs1459425933	26	dbSNP
rs746466106	26	dbSNP
rs777801803	28	dbSNP
rs1252109926	30	dbSNP
rs745700830	32	dbSNP
rs556292374	33	dbSNP
rs775541797	34	dbSNP
rs374425918	35	dbSNP
rs879302835	36	dbSNP
rs1248523654	39	dbSNP
rs1474796450	40	dbSNP
rs768335637	41	dbSNP
rs776272824	43	dbSNP
rs771471821	46	dbSNP
rs1405561139	49	dbSNP
rs761337910	51	dbSNP
rs1166164114	52	dbSNP
rs764952437	53	dbSNP
rs1204342650	55	dbSNP
rs1251046255	57	dbSNP
rs1455125774	60	dbSNP
rs1386248910	62	dbSNP
rs1157894130	63	dbSNP
rs1420631710	64	dbSNP
rs577870677	65	dbSNP
rs951258485	70	dbSNP
rs1263002194	71	dbSNP
rs1188847630	76	dbSNP
rs1376673071	78	dbSNP
rs545243995	87	dbSNP
rs1174908296	89	dbSNP
rs977629097	90	dbSNP
rs924920851	94	dbSNP
rs1369253161	95	dbSNP
rs969216082	98	dbSNP
rs933313428	105	dbSNP
rs1429748626	107	dbSNP
rs375901508	108	dbSNP
rs980599033	119	dbSNP
rs117603140	123	dbSNP
rs1437219134	128	dbSNP
rs1362720090	130	dbSNP
rs766880543	131	dbSNP
rs191816327	134	dbSNP
rs927400221	135	dbSNP
rs1392419022	136	dbSNP
rs1161134935	137	dbSNP
rs957637590	139	dbSNP
rs943408026	149	dbSNP
rs990088172	156	dbSNP
rs767453916	169	dbSNP
rs1173110056	175	dbSNP
rs1385423564	182	dbSNP
rs1247732557	187	dbSNP
rs913209041	189	dbSNP
rs1037809808	197	dbSNP
rs142963445	203	dbSNP
rs1236605839	217	dbSNP
rs374640411	236	dbSNP
rs898315111	238	dbSNP
rs1253180612	239	dbSNP
rs531400511	240	dbSNP
rs1339992793	246	dbSNP
rs1025976177	256	dbSNP
rs550903756	259	dbSNP
rs887071327	260	dbSNP
rs1332658108	266	dbSNP
rs569385296	267	dbSNP
rs533358210	272	dbSNP
rs1385902121	278	dbSNP
rs912739539	282	dbSNP
rs942875555	283	dbSNP
rs1039898967	284	dbSNP
rs1207790679	288	dbSNP
rs182447049	289	dbSNP
rs369871528	295	dbSNP
rs1174859702	298	dbSNP
rs1207195921	307	dbSNP
rs1035333726	312	dbSNP
rs1198379188	313	dbSNP
rs1376308265	313	dbSNP
rs1252692952	323	dbSNP
rs898225925	323	dbSNP
rs764673929	329	dbSNP
rs150704948	340	dbSNP
rs1046777378	342	dbSNP
rs886750347	342	dbSNP
rs1012600321	347	dbSNP
rs1021358090	348	dbSNP
rs879117598	349	dbSNP
rs1387846455	350	dbSNP
rs1448326579	351	dbSNP
rs1171785522	352	dbSNP
rs1375249955	356	dbSNP
rs1456736800	357	dbSNP
rs1013509983	361	dbSNP
rs533957463	366	dbSNP
rs1024862750	376	dbSNP
rs969659048	384	dbSNP
rs1407417587	387	dbSNP
rs1369456043	390	dbSNP
rs1002039638	398	dbSNP
rs1416685016	401	dbSNP
rs1400428572	402	dbSNP
rs1172379789	403	dbSNP
rs1395662133	410	dbSNP
rs750017537	418	dbSNP
rs749890809	420	dbSNP
rs957492096	423	dbSNP
rs1318755700	424	dbSNP
rs1472276238	431	dbSNP
rs1258352847	435	dbSNP
rs1211421111	436	dbSNP
rs990171936	439	dbSNP
rs760005949	440	dbSNP
rs1296233762	441	dbSNP
rs186808029	441	dbSNP
rs1309123681	442	dbSNP
rs977597799	445	dbSNP
rs1289807800	447	dbSNP
rs567439394	457	dbSNP
rs924774070	458	dbSNP
rs954948658	463	dbSNP
rs1226215526	465	dbSNP
rs1335223385	467	dbSNP
rs1293380105	472	dbSNP
rs1450741252	474	dbSNP
rs1339218772	476	dbSNP
rs140703579	481	dbSNP
rs913240042	486	dbSNP
rs1401621748	488	dbSNP
rs555416426	491	dbSNP
rs1157161787	492	dbSNP
rs1456916234	494	dbSNP
rs987048401	495	dbSNP
rs577816390	497	dbSNP
rs942886349	499	dbSNP
rs192269945	502	dbSNP
rs1235633855	503	dbSNP
rs943375448	506	dbSNP
rs763850902	514	dbSNP
rs1037777936	516	dbSNP
rs920712514	517	dbSNP
rs1039860055	518	dbSNP
rs1328065059	522	dbSNP
rs920059482	528	dbSNP
rs1229537062	529	dbSNP
rs1376395143	530	dbSNP
rs931095206	531	dbSNP
rs1013248793	532	dbSNP
rs1046807252	532	dbSNP
rs375632115	532	dbSNP
rs886780097	532	dbSNP
rs753431745	545	dbSNP
rs1186547387	552	dbSNP
rs1434898959	552	dbSNP
rs1231731438	555	dbSNP
rs931132453	567	dbSNP
rs543610464	580	dbSNP
rs1413192590	585	dbSNP
rs1046384340	587	dbSNP
rs905088128	587	dbSNP
rs1046854832	588	dbSNP
rs554142404	589	dbSNP
rs758725978	591	dbSNP
rs572348679	594	dbSNP
rs879744296	595	dbSNP
rs1463311467	596	dbSNP
rs1157257872	598	dbSNP
rs1388121162	599	dbSNP
rs1431587110	600	dbSNP
rs1020319301	601	dbSNP
rs1290638111	602	dbSNP
rs1377063969	603	dbSNP
rs894130590	611	dbSNP
rs34221783	627	dbSNP
rs1012569545	629	dbSNP
rs1344914478	630	dbSNP
rs1296131523	632	dbSNP
rs1228085125	638	dbSNP
rs866853410	639	dbSNP
rs1372241250	657	dbSNP
rs1021325362	666	dbSNP
rs377726605	668	dbSNP
rs542659635	671	dbSNP
rs1020315068	672	dbSNP
rs1355896813	675	dbSNP
rs1329639174	682	dbSNP
rs1234867245	684	dbSNP
rs1431769034	686	dbSNP
rs1265883774	687	dbSNP
rs1468154299	689	dbSNP
rs1166691149	695	dbSNP
rs1448623844	697	dbSNP
rs1425639506	698	dbSNP
rs1315903172	701	dbSNP
rs1487307314	706	dbSNP
rs1245823744	729	dbSNP
rs999016446	730	dbSNP
rs964339462	732	dbSNP
rs373113534	735	dbSNP
rs1228340624	737	dbSNP
rs920108051	737	dbSNP
rs10092194	750	dbSNP
rs1258776715	762	dbSNP
rs987723987	766	dbSNP
rs1367155841	768	dbSNP
rs543549219	769	dbSNP
rs778207826	772	dbSNP
rs1407532094	774	dbSNP
rs1306057484	775	dbSNP
rs1429069059	777	dbSNP
rs567302268	789	dbSNP
rs973442352	794	dbSNP
rs36107033	796	dbSNP
rs1177947556	797	dbSNP
rs1161540457	798	dbSNP
rs1431291394	800	dbSNP
rs1046050582	801	dbSNP
rs144579109	802	dbSNP
rs867670257	804	dbSNP
rs868131644	809	dbSNP
rs1483873471	811	dbSNP
rs1355964348	815	dbSNP
rs1258347645	817	dbSNP
rs367856231	821	dbSNP
rs908353027	827	dbSNP
rs551658308	833	dbSNP
rs1372343668	836	dbSNP
rs1224038876	845	dbSNP
rs1009354342	846	dbSNP
rs1020350427	848	dbSNP
rs1209462574	850	dbSNP
rs1057039363	854	dbSNP
rs1379795173	865	dbSNP
rs1304550834	872	dbSNP
rs1008237870	881	dbSNP
rs1019629832	884	dbSNP
rs1281682241	886	dbSNP
rs1409056648	895	dbSNP
rs894434779	897	dbSNP
rs1180812155	915	dbSNP
rs796682507	924	dbSNP
rs566842218	925	dbSNP
rs1486900783	935	dbSNP
rs1180679058	936	dbSNP
rs867051844	942	dbSNP
rs1203624750	944	dbSNP
rs1232724029	945	dbSNP
rs964350330	960	dbSNP
rs1012787117	962	dbSNP
rs1470704934	966	dbSNP
rs1179213767	978	dbSNP
rs1267810918	981	dbSNP
rs1042665844	985	dbSNP
rs1323631086	997	dbSNP
rs904208867	1004	dbSNP
rs781123779	1005	dbSNP
rs527948576	1006	dbSNP
rs745981399	1009	dbSNP
rs1320995341	1014	dbSNP
rs1437082825	1016	dbSNP
rs747075217	1016	dbSNP
rs1385992844	1020	dbSNP
rs890617304	1026	dbSNP
rs1457952262	1032	dbSNP
rs548977103	1037	dbSNP
rs1009515265	1038	dbSNP
rs1017868230	1045	dbSNP
rs1423752614	1046	dbSNP
rs567277573	1048	dbSNP
rs964920666	1049	dbSNP
rs1306080710	1050	dbSNP
rs1463852679	1053	dbSNP
rs142705003	1055	dbSNP
rs1247613639	1058	dbSNP
rs550292133	1062	dbSNP
rs908318951	1064	dbSNP
rs1299543991	1068	dbSNP
rs1364980371	1069	dbSNP
rs1027628913	1070	dbSNP
rs950801014	1073	dbSNP
rs949136170	1074	dbSNP
rs1298324362	1075	dbSNP
rs572589965	1078	dbSNP
rs1230301849	1079	dbSNP
rs1362828510	1084	dbSNP
rs982231617	1085	dbSNP
rs571635737	1092	dbSNP
rs926319129	1099	dbSNP
rs1291303818	1112	dbSNP
rs1423521547	1114	dbSNP
rs1391287743	1115	dbSNP
rs1349400288	1128	dbSNP
rs750931279	1140	dbSNP
rs1447990790	1145	dbSNP
rs538855592	1149	dbSNP
rs1389979432	1151	dbSNP
rs983543421	1153	dbSNP
rs928071761	1157	dbSNP
rs1438787110	1158	dbSNP
rs1053748970	1161	dbSNP
rs1207006593	1167	dbSNP
rs1245219341	1172	dbSNP
rs1468155020	1173	dbSNP
rs1194502852	1176	dbSNP
rs1397033329	1177	dbSNP
rs1275239791	1179	dbSNP
rs1207241925	1180	dbSNP
rs1339760700	1184	dbSNP
rs893707422	1186	dbSNP
rs938525096	1188	dbSNP
rs1332454978	1189	dbSNP
rs1298787597	1192	dbSNP
rs1166115899	1196	dbSNP
rs1370655848	1198	dbSNP
rs1310346488	1199	dbSNP
rs992580043	1200	dbSNP
rs915752091	1206	dbSNP
rs948608141	1207	dbSNP
rs1410333207	1208	dbSNP
rs1042970170	1217	dbSNP
rs1173511217	1221	dbSNP
rs890263063	1224	dbSNP
rs1376101027	1226	dbSNP
rs758859288	1227	dbSNP
rs1330560109	1229	dbSNP
rs553981316	1232	dbSNP
rs1472932025	1236	dbSNP
rs1128779	1240	dbSNP
rs1392008962	1248	dbSNP
rs879602554	1252	dbSNP
rs1184174703	1256	dbSNP
rs1439778973	1257	dbSNP
rs1482043029	1260	dbSNP
rs1041491309	1265	dbSNP
rs1200784806	1268	dbSNP
rs572379302	1277	dbSNP
rs997250673	1278	dbSNP
rs1222556221	1280	dbSNP
rs934352890	1284	dbSNP
rs751968586	1285	dbSNP
rs1432213704	1293	dbSNP
rs1052867150	1305	dbSNP
rs890242822	1313	dbSNP
rs1335683726	1325	dbSNP
rs1004436969	1330	dbSNP
rs1216699505	1332	dbSNP
rs1288261271	1338	dbSNP
rs1015775953	1340	dbSNP

- Nature, 2009

MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein-RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein-RNA complexes in mouse brain. This produced two simultaneous data sets-Ago-miRNA and Ago-mRNA binding sites-that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA-mRNA interactions.

- Genes & development, 2013

When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3' untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response.

- Breast cancer research and treatment, 2014

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

- Nucleic acids research, 2016

MicroRNAs (miRs) have emerged as key biological effectors in human health and disease. These small noncoding RNAs are incorporated into Argonaute (Ago) proteins, where they direct post-transcriptional gene silencing via base-pairing with target transcripts. Although miRs have become intriguing biological entities and attractive therapeutic targets, the translational impacts of miR research remain limited by a paucity of empirical miR targeting data, particularly in human primary tissues. Here, to improve our understanding of the diverse roles miRs play in cardiovascular function and disease, we applied high-throughput methods to globally profile miR:target interactions in human heart tissues. We deciphered Ago2:RNA interactions using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Our initial exploration of this interactome revealed an abundance of miR target sites in gene coding regions, including several sites pointing to new miR-29 functions in regulating cardiomyocyte calcium, growth and metabolism. Also, we uncovered several clinically-relevant interactions involving common genetic variants that alter miR targeting events in cardiomyopathy-associated genes. Overall, these data provide a critical resource for bolstering translational miR research in heart, and likely beyond.

- Molecular cell, 2017

MicroRNA-122, an abundant and conserved liver-specific miRNA, regulates hepatic metabolism and functions as a tumor suppressor, yet systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Argonaute crosslinking immunoprecipitation (Argonaute [Ago]-CLIP) sequencing in miR-122 knockout and control mouse livers, as well as in matched human hepatocellular carcinoma (HCC) and benign liver tissue to identify miRNA target sites transcriptome-wide in two species. We observed a majority of miR-122 binding on 3' UTRs and coding exons followed by extensive binding to other genic and non-genic sites. Motif analysis of miR-122-dependent binding revealed a G-bulged motif in addition to canonical motifs. A large number of miR-122 targets were found to be species specific. Upregulation of several common mouse and human targets, most notably BCL9, predicted survival in HCC patients. These results broadly define the molecular consequences of miR-122 downregulation in hepatocellular carcinoma.