ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uacGUCACCUG---GUCCUACCGUUc 5' | | |||| |||:|||||: Target 5' gtcCTGGGGACGTGGAGGGTGGCAGc 3'	643 - 668	132.00	-19.70
2	miRNA 3' uacGUC--ACCUGGUCCUACCGUUc 5' ||| || : || | |||||| Target 5' cccCAGAATGAGGCA-GTTGGCAAa 3'	390 - 413	125.00	-11.80
3	miRNA 3' uaCGUCACCUGGUCCUACCGUuc 5' |::|||| || ||||| Target 5' gtGTGGTGGCCC----TGGCAct 3'	81 - 99	114.00	-15.10

Mutant ID	Mutant Position	Mutant Source
COSN18729340	4	COSMIC
COSN30120110	8	COSMIC
COSN24384263	38	COSMIC
COSN29735125	163	COSMIC
COSN21522570	173	COSMIC
COSN15663077	359	COSMIC
COSN16155822	361	COSMIC
COSN17413524	522	COSMIC
COSN31531424	609	COSMIC
COSN30176898	643	COSMIC
COSN26550754	673	COSMIC
COSN25125502	689	COSMIC
COSN6793896	743	COSMIC
COSN31609141	771	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1422330706	1	dbSNP
rs752660628	3	dbSNP
rs968456186	4	dbSNP
rs1460790640	5	dbSNP
rs552180452	5	dbSNP
rs1376031386	6	dbSNP
rs1172734455	11	dbSNP
rs1019998811	12	dbSNP
rs1391827412	13	dbSNP
rs1171393348	14	dbSNP
rs767750268	16	dbSNP
rs1417385069	17	dbSNP
rs1183702998	21	dbSNP
rs375089554	23	dbSNP
rs1257406162	24	dbSNP
rs865862007	26	dbSNP
rs371763271	27	dbSNP
rs1252312285	30	dbSNP
rs766641046	32	dbSNP
rs998478862	33	dbSNP
rs763015951	34	dbSNP
rs1334595529	35	dbSNP
rs368867475	36	dbSNP
rs1045466	37	dbSNP
rs1234622210	43	dbSNP
rs1005969919	44	dbSNP
rs551713236	51	dbSNP
rs115317882	53	dbSNP
rs1418830378	54	dbSNP
rs1047212435	61	dbSNP
rs1045469	79	dbSNP
rs1268746803	86	dbSNP
rs929792169	91	dbSNP
rs773751101	92	dbSNP
rs541511602	95	dbSNP
rs907349261	97	dbSNP
rs1057374943	112	dbSNP
rs937249430	113	dbSNP
rs529846447	115	dbSNP
rs927203620	118	dbSNP
rs978415667	128	dbSNP
rs947087201	129	dbSNP
rs1451818872	135	dbSNP
rs1045470	136	dbSNP
rs887794181	141	dbSNP
rs1049051514	142	dbSNP
rs1361236258	149	dbSNP
rs562305900	151	dbSNP
rs1236694612	152	dbSNP
rs988540959	156	dbSNP
rs956858198	157	dbSNP
rs1229425802	158	dbSNP
rs1036946943	163	dbSNP
rs1029804010	164	dbSNP
rs765714654	167	dbSNP
rs1388454871	169	dbSNP
rs1284954515	170	dbSNP
rs1218230105	171	dbSNP
rs1255285366	173	dbSNP
rs964290027	175	dbSNP
rs1018951262	176	dbSNP
rs1257926970	180	dbSNP
rs1005544211	181	dbSNP
rs1421556763	182	dbSNP
rs888488723	185	dbSNP
rs1302016766	188	dbSNP
rs1358678602	192	dbSNP
rs1025716676	194	dbSNP
rs1449323864	197	dbSNP
rs1305118102	200	dbSNP
rs181483701	201	dbSNP
rs895921251	203	dbSNP
rs1156441722	206	dbSNP
rs1377238695	209	dbSNP
rs1453573198	210	dbSNP
rs1277493844	219	dbSNP
rs1311104653	220	dbSNP
rs932379883	222	dbSNP
rs1378589819	223	dbSNP
rs765360395	225	dbSNP
rs989742813	239	dbSNP
rs955726735	242	dbSNP
rs544119555	245	dbSNP
rs1056926063	247	dbSNP
rs1424465546	248	dbSNP
rs1032361833	253	dbSNP
rs937177832	256	dbSNP
rs1353322752	259	dbSNP
rs905770395	260	dbSNP
rs576753551	264	dbSNP
rs1332418934	268	dbSNP
rs1379585923	272	dbSNP
rs1043403735	275	dbSNP
rs1023113942	278	dbSNP
rs1366268640	279	dbSNP
rs4974572	291	dbSNP
rs1219355070	294	dbSNP
rs1303275589	304	dbSNP
rs1194526526	307	dbSNP
rs1258403830	309	dbSNP
rs947033471	311	dbSNP
rs1489307838	318	dbSNP
rs912930939	320	dbSNP
rs1212053551	325	dbSNP
rs1013008678	327	dbSNP
rs887828831	334	dbSNP
rs1204784810	337	dbSNP
rs376995762	341	dbSNP
rs1027731924	342	dbSNP
rs993944421	343	dbSNP
rs190712719	349	dbSNP
rs1037386363	350	dbSNP
rs1460257182	351	dbSNP
rs923017738	354	dbSNP
rs1321148492	357	dbSNP
rs374555956	359	dbSNP
rs1337792000	360	dbSNP
rs1436790168	360	dbSNP
rs909805634	360	dbSNP
rs1045480	361	dbSNP
rs1413822417	361	dbSNP
rs1218782310	362	dbSNP
rs1341820916	363	dbSNP
rs1452367395	363	dbSNP
rs943940976	364	dbSNP
rs890972667	365	dbSNP
rs555048849	369	dbSNP
rs1426470334	371	dbSNP
rs1474412245	371	dbSNP
rs1162996133	372	dbSNP
rs1415006173	373	dbSNP
rs1234269129	375	dbSNP
rs1358070933	375	dbSNP
rs937690023	375	dbSNP
rs1298711383	377	dbSNP
rs1305009007	377	dbSNP
rs186984898	378	dbSNP
rs1429286212	379	dbSNP
rs569490067	383	dbSNP
rs1171989752	385	dbSNP
rs1479053643	386	dbSNP
rs1371408927	387	dbSNP
rs1192516600	390	dbSNP
rs932413967	394	dbSNP
rs1217486054	403	dbSNP
rs1265464381	408	dbSNP
rs1484948173	428	dbSNP
rs976914355	433	dbSNP
rs140581613	436	dbSNP
rs964595256	438	dbSNP
rs911475152	439	dbSNP
rs11540633	445	dbSNP
rs76532252	449	dbSNP
rs572008054	459	dbSNP
rs1459810395	461	dbSNP
rs751473672	461	dbSNP
rs914358719	465	dbSNP
rs1262494083	470	dbSNP
rs1164626512	478	dbSNP
rs1378153918	482	dbSNP
rs1418530703	486	dbSNP
rs989857482	487	dbSNP
rs1361927105	490	dbSNP
rs1319825446	501	dbSNP
rs1412631578	502	dbSNP
rs77379446	507	dbSNP
rs1267920133	508	dbSNP
rs934348061	515	dbSNP
rs1450011076	517	dbSNP
rs924270159	519	dbSNP
rs984396054	522	dbSNP
rs1376384180	523	dbSNP
rs1241177380	524	dbSNP
rs952953013	526	dbSNP
rs982116635	535	dbSNP
rs1226028502	540	dbSNP
rs1342966961	543	dbSNP
rs1200381940	544	dbSNP
rs1340739576	545	dbSNP
rs1444468040	550	dbSNP
rs1025626835	551	dbSNP
rs563184205	553	dbSNP
rs527281344	554	dbSNP
rs564855534	556	dbSNP
rs566302187	570	dbSNP
rs1036216618	572	dbSNP
rs372329092	573	dbSNP
rs993584251	580	dbSNP
rs1416132091	583	dbSNP
rs1328982130	585	dbSNP
rs1399290585	590	dbSNP
rs962366565	609	dbSNP
rs147222661	610	dbSNP
rs1008565041	615	dbSNP
rs879622540	617	dbSNP
rs905713261	619	dbSNP
rs1181965351	620	dbSNP
rs1347708990	622	dbSNP
rs1042963534	625	dbSNP
rs1011421493	626	dbSNP
rs1257763517	627	dbSNP
rs772183606	629	dbSNP
rs1198864620	630	dbSNP
rs1252285589	636	dbSNP
rs1430916890	637	dbSNP
rs529712088	638	dbSNP
rs932997024	639	dbSNP
rs533092925	643	dbSNP
rs934401482	646	dbSNP
rs1242307904	647	dbSNP
rs1168448771	650	dbSNP
rs574402308	651	dbSNP
rs1439540178	653	dbSNP
rs1045369986	654	dbSNP
rs1333580978	656	dbSNP
rs1039235631	657	dbSNP
rs544035199	659	dbSNP
rs911409145	660	dbSNP
rs916204848	667	dbSNP
rs1356420165	668	dbSNP
rs926283257	669	dbSNP
rs771747026	671	dbSNP
rs1381532957	672	dbSNP
rs920733784	673	dbSNP
rs1487279454	674	dbSNP
rs952901006	675	dbSNP
rs972576408	678	dbSNP
rs1267370195	680	dbSNP
rs1490714222	683	dbSNP
rs532166377	689	dbSNP
rs565230395	690	dbSNP
rs1478012794	695	dbSNP
rs1172202507	698	dbSNP
rs1310472454	701	dbSNP
rs1449440360	704	dbSNP
rs1457533814	705	dbSNP
rs962113791	715	dbSNP
rs1392950288	722	dbSNP
rs1019373610	744	dbSNP
rs1298104485	746	dbSNP
rs960353672	754	dbSNP
rs953378588	771	dbSNP
rs1310945681	774	dbSNP
rs1035758218	780	dbSNP
rs1028221986	782	dbSNP
rs996688179	789	dbSNP
rs1213887599	794	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.

- 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.