ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ccUUUGUCCUUUUUUGUAa 5' :|| || ||:||||| Target 5' tgGAAAAGTTAAGAACATt 3'	235 - 253	141.00	-10.80
2	miRNA 3' ccUUUGUCCUUUUUUGUAa 5' |||: |:||:||:|| Target 5' ccAAAT--GGAAGAATATt 3'	739 - 755	125.00	-6.20
3	miRNA 3' ccUUUGUCCUU-----UUUUGUAa 5' ||:|||||| ||:|| | Target 5' ggAAGCAGGAAGGCCCAAGACCTg 3'	8 - 31	117.00	-15.29

Mutant ID	Mutant Position	Mutant Source
COSN30450073	15	COSMIC
COSN31587435	20	COSMIC
COSN30179598	38	COSMIC
COSN31492786	39	COSMIC
COSN19003373	61	COSMIC
COSN6035151	122	COSMIC
COSN15720035	138	COSMIC
COSN31522777	147	COSMIC
COSN31595149	186	COSMIC
COSN31959412	679	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1474327672	6	dbSNP
rs745626718	9	dbSNP
rs757033765	20	dbSNP
rs753760956	29	dbSNP
rs116663798	32	dbSNP
rs755668390	38	dbSNP
rs752319475	41	dbSNP
rs767044057	45	dbSNP
rs912033196	56	dbSNP
rs987524394	61	dbSNP
rs932029497	62	dbSNP
rs1452491664	76	dbSNP
rs921978285	92	dbSNP
rs1342600914	93	dbSNP
rs1225777171	102	dbSNP
rs1327602565	113	dbSNP
rs1269212139	116	dbSNP
rs979164835	118	dbSNP
rs752985444	120	dbSNP
rs543992314	127	dbSNP
rs1260660027	131	dbSNP
rs187139243	138	dbSNP
rs955625138	139	dbSNP
rs1220357565	140	dbSNP
rs1366966292	142	dbSNP
rs1273173319	148	dbSNP
rs1445055618	156	dbSNP
rs1192132716	169	dbSNP
rs1433101866	179	dbSNP
rs1389658069	180	dbSNP
rs1022617149	183	dbSNP
rs1427269850	184	dbSNP
rs988049650	185	dbSNP
rs1177776867	188	dbSNP
rs975238156	190	dbSNP
rs1381034763	212	dbSNP
rs956536187	213	dbSNP
rs1034816210	216	dbSNP
rs541641405	217	dbSNP
rs1022142099	221	dbSNP
rs1456496310	230	dbSNP
rs1281022370	234	dbSNP
rs1003294692	237	dbSNP
rs969123074	242	dbSNP
rs766213294	255	dbSNP
rs1223658805	256	dbSNP
rs1023332530	273	dbSNP
rs1178103379	281	dbSNP
rs1433725898	290	dbSNP
rs570641323	292	dbSNP
rs1196070959	294	dbSNP
rs1267114931	297	dbSNP
rs1199596033	299	dbSNP
rs1480141309	299	dbSNP
rs1379447902	303	dbSNP
rs1188958074	304	dbSNP
rs994897894	315	dbSNP
rs529826597	319	dbSNP
rs956661899	322	dbSNP
rs1421978976	323	dbSNP
rs1031058967	325	dbSNP
rs1300890004	325	dbSNP
rs1405185383	331	dbSNP
rs559251123	339	dbSNP
rs1302716036	343	dbSNP
rs182505472	344	dbSNP
rs577209437	347	dbSNP
rs1261287893	352	dbSNP
rs1355574215	353	dbSNP
rs1220047971	359	dbSNP
rs1290773577	398	dbSNP
rs899438037	403	dbSNP
rs745489469	406	dbSNP
rs1005057484	414	dbSNP
rs1417456634	416	dbSNP
rs190483363	425	dbSNP
rs890627651	426	dbSNP
rs1051877697	427	dbSNP
rs1442100798	429	dbSNP
rs932104720	431	dbSNP
rs1365381107	436	dbSNP
rs1023375528	445	dbSNP
rs1013036432	455	dbSNP
rs894608146	456	dbSNP
rs1038542575	459	dbSNP
rs1307598086	461	dbSNP
rs1378942155	471	dbSNP
rs941585311	472	dbSNP
rs887391056	473	dbSNP
rs543770485	474	dbSNP
rs1043088330	475	dbSNP
rs1222555444	479	dbSNP
rs947395098	484	dbSNP
rs913200636	489	dbSNP
rs1479691266	490	dbSNP
rs988468534	493	dbSNP
rs576474350	522	dbSNP
rs927820060	525	dbSNP
rs1459453861	530	dbSNP
rs758302036	531	dbSNP
rs1384981976	532	dbSNP
rs969154193	537	dbSNP
rs1433218159	539	dbSNP
rs776394949	546	dbSNP
rs770513640	547	dbSNP
rs1166473445	550	dbSNP
rs1362709203	550	dbSNP
rs1023786803	551	dbSNP
rs1371039276	554	dbSNP
rs1466426779	555	dbSNP
rs1461182059	556	dbSNP
rs774646946	556	dbSNP
rs558163376	557	dbSNP
rs1173942699	559	dbSNP
rs1431104382	562	dbSNP
rs975457672	562	dbSNP
rs1336059176	563	dbSNP
rs942771901	566	dbSNP
rs1167550458	569	dbSNP
rs1447304501	572	dbSNP
rs555085335	579	dbSNP
rs1182806562	584	dbSNP
rs1483174503	600	dbSNP
rs963382994	616	dbSNP
rs1208004357	619	dbSNP
rs765050575	627	dbSNP
rs1258736943	629	dbSNP
rs1320153041	631	dbSNP
rs1202293893	638	dbSNP
rs1243135705	641	dbSNP
rs75464904	642	dbSNP
rs530024611	643	dbSNP
rs1260094566	659	dbSNP
rs1226409493	660	dbSNP
rs572671331	661	dbSNP
rs890702140	663	dbSNP
rs989345311	667	dbSNP
rs956813819	668	dbSNP
rs1175614245	669	dbSNP
rs1359612910	674	dbSNP
rs1314540515	676	dbSNP
rs1396382336	677	dbSNP
rs1030943957	678	dbSNP
rs7686	679	dbSNP
rs1279122041	684	dbSNP
rs1320084961	685	dbSNP
rs1484731347	686	dbSNP
rs184228046	697	dbSNP
rs1313235170	698	dbSNP
rs1212902883	710	dbSNP
rs1411642799	711	dbSNP
rs181554343	718	dbSNP
rs1167447571	722	dbSNP
rs1479259550	725	dbSNP
rs900647268	730	dbSNP
rs1011917004	732	dbSNP
rs1416049528	736	dbSNP
rs1043545790	742	dbSNP
rs947458297	743	dbSNP
rs1420145933	754	dbSNP
rs12395	761	dbSNP
rs148951967	768	dbSNP
rs1342550508	771	dbSNP
rs1412130483	771	dbSNP
rs1053104251	775	dbSNP
rs935268935	776	dbSNP
rs1368428346	780	dbSNP
rs1184941443	789	dbSNP
rs375505058	790	dbSNP
rs1005748565	791	dbSNP
rs1316693889	792	dbSNP
rs1217880947	797	dbSNP
rs1047801	799	dbSNP
rs14614	800	dbSNP
rs981965300	801	dbSNP
rs1047804	802	dbSNP
rs778978467	802	dbSNP
rs1221308955	803	dbSNP
rs1203130679	806	dbSNP
rs969645725	807	dbSNP
rs1489540504	809	dbSNP
rs1175405296	819	dbSNP
rs540166672	823	dbSNP
rs916382666	833	dbSNP
rs1320870420	838	dbSNP
rs147311863	838	dbSNP
rs973564649	844	dbSNP
rs963158181	850	dbSNP
rs1230106332	865	dbSNP
rs1328289768	867	dbSNP
rs141678587	868	dbSNP
rs1350511868	869	dbSNP
rs942737484	872	dbSNP
rs1278281712	883	dbSNP
rs1306808326	889	dbSNP
rs983067968	889	dbSNP
rs796491658	892	dbSNP
rs954984167	897	dbSNP
rs1392338521	900	dbSNP
rs989704999	909	dbSNP
rs1227350467	913	dbSNP
rs1293720326	914	dbSNP
rs1271402911	917	dbSNP
rs1452816743	917	dbSNP
rs935211102	920	dbSNP
rs1231821738	921	dbSNP
rs1457136608	936	dbSNP
rs1458130199	936	dbSNP
rs529885137	937	dbSNP
rs1161832137	940	dbSNP
rs1473171954	941	dbSNP
rs10015	942	dbSNP
rs377551518	960	dbSNP
rs559113176	960	dbSNP
rs71671712	960	dbSNP
rs1407308303	963	dbSNP
rs916218412	973	dbSNP
rs1399673102	979	dbSNP
rs900673780	981	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.

- 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.

- 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.