>TSC22D3|NM_198057|3'UTR
   1 GTGGCTCTGTCCTCAGGGTGGGCAGAGCCACTAAACTTGTTTTACCTAGTTCTTTCCAGTTTGTTTTTGGCTCCCCAAGC
  81 ATCATCTCACGAGGAGAACTTTACACCTAGCACAGCTGGTGCCAAGAGATGTCCTAAGGACATGGCCACCTGGGTCCACT
 161 CCAGCGACAGACCCCTGACAAGAGCAGGTCTCTGGAGGCTGAGTTGCATGGGGCCTAGTAACACCAAGCCAGTGAGCCTC
 241 TAATGCTACTGCGCCCTGGGGGCTCCCAGGGCCTGGGCAACTTAGCTGCAACTGGCAAAGGAGAAGGGTAGTTTGAGGTG
 321 TGACACCAGTTTGCTCCAGAAAGTTTAAGGGGTCTGTTTCTCATCTCCATGGACATCTTCAACAGCTTCACCTGACAACG
 401 ACTGTTCCTATGAAGAAGCCACTTGTGTTTTAAGCAGAGGCAACCTCTCTCTTCTCCTCTGTTTCGTGAAGGCAGGGGAC
 481 ACAGATGGGAGAGATTGAGCCAAGTCAGCCTTCTGTTGGTTAATATGGTATAATGCATGGCTTTGTGCACAGCCCAGTGT
 561 GGGATTACAGCTTTGGGATGACCGCTTACAAAGTTCTGTTTGGTTAGTATTGGCATAGTTTTTCTATATAGCCATAAATG
 641 CGTATATATACCCATAGGGCTAGATCTGTATCTTAGTGTAGCGATGTATACATATACACATCCACCTACATGTTGAAGGG
 721 CCTAACCAGCCTTGGGAGTATTGACTGGTCCCTTACCTCTTATGGCTAAGTCTTTGACTGTGTTCATTTACCAAGTTGAC
 801 CCAGTTTGTCTTTTAGGTTAAGTAAGACTCGAGAGTAAAGGCAAGGAGGGGGGCCAGCCTCTGAATGCGGCCACGGATGC
 881 CTTGCTGCTGCAACCCTTTCCCCAGCTGTCCACTGAAACGTGAAGTCCTGTTTTGAATGCCAAACCCACCATTCACTGGT
 961 GCTGACTACATAGAATGGGGTTGAGAGAAGATCAGTTTGGGCTTCACAGTGTCATTTGAAAACGTTTTTTGTTTTGTTTT
1041 GTAATTATTGTGGAAAACTTTCAAGTGAACAGAAGGATGGTGTCCTACTGTGGATGAGGGATGAACAAGGGGATGGCTTT
1121 GATCCAATGGAGCCTGGGAGGTGTGCCCAGAAAGCTTGTCTGTAGCGGGTTTTGTGAGAGTGAACACTTTCCACTTTTTG
1201 ACACCTTATCCTGATGTATGGTTCCAGGATTTGGATTTTGATTTTCCAAATGTAGCTTGAAATTTCAATAAACTTTGCTC
1281 TGTTTTTCTAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucGUGUCUGA--ACG--ACACUACAa 5' ||||| || ||| | ||||| Target 5' agCACAG-CTGGTGCCAAGAGATGTc 3'	109 - 133	127.00	-14.20
2	miRNA 3' ucgUGUCUGAACGACACUACAa 5' ||| ||| : |||||| Target 5' ttgACA-CCTTATCCTGATGTa 3'	1198 - 1218	122.00	-6.50
3	miRNA 3' ucGUGUCUGAACGACACUACAa 5' | :|| ||: | ||||| Target 5' atCTTAG---TGTAGCGATGTa 3'	670 - 688	117.00	-6.20

Mutant ID	Mutant Position	Mutant Source
rs1275816911	4	dbSNP
rs1204187005	5	dbSNP
rs1017346716	8	dbSNP
rs761271427	16	dbSNP
rs773556062	18	dbSNP
rs772604324	19	dbSNP
rs1377421531	20	dbSNP
rs748514130	23	dbSNP
rs1411290120	27	dbSNP
rs1350651982	29	dbSNP
rs774692162	33	dbSNP
rs1439739278	36	dbSNP
rs1452436659	46	dbSNP
rs1001650552	47	dbSNP
rs1412650462	51	dbSNP
rs1297503687	64	dbSNP
rs1326927048	70	dbSNP
rs1243855119	76	dbSNP
rs903807688	83	dbSNP
rs1249552098	84	dbSNP
rs758212082	90	dbSNP
rs1212247995	91	dbSNP
rs773909537	104	dbSNP
rs945252394	106	dbSNP
rs1315674228	116	dbSNP
rs771280473	118	dbSNP
rs1484511086	126	dbSNP
rs189584221	129	dbSNP
rs1262944426	141	dbSNP
rs1045584129	149	dbSNP
rs944100953	150	dbSNP
rs1356260886	157	dbSNP
rs1454725381	174	dbSNP
rs1173122134	186	dbSNP
rs1373834722	188	dbSNP
rs949865698	191	dbSNP
rs1290397857	215	dbSNP
rs1311064864	222	dbSNP
rs1381455308	249	dbSNP
rs891718565	253	dbSNP
rs1373139885	261	dbSNP
rs764613224	262	dbSNP
rs985391454	300	dbSNP
rs1345280787	307	dbSNP
rs1381540078	310	dbSNP
rs12689231	325	dbSNP
rs1240831289	334	dbSNP
rs1260949145	340	dbSNP
rs1485711496	351	dbSNP
rs1203776315	352	dbSNP
rs1302927035	353	dbSNP
rs1469337402	361	dbSNP
rs1193062514	369	dbSNP
rs1052932139	377	dbSNP
rs1449517570	382	dbSNP
rs1393409672	391	dbSNP
rs778279639	399	dbSNP
rs770407980	400	dbSNP
rs1363464535	404	dbSNP
rs775650103	409	dbSNP
rs974793168	410	dbSNP
rs1452736728	415	dbSNP
rs920214250	428	dbSNP
rs943161852	429	dbSNP
rs765303619	454	dbSNP
rs1441625384	460	dbSNP
rs374793446	465	dbSNP
rs987004835	466	dbSNP
rs1224977766	472	dbSNP
rs974719596	495	dbSNP
rs1315995898	508	dbSNP
rs1237291453	509	dbSNP
rs1275495029	526	dbSNP
rs961582459	536	dbSNP
rs1213693176	549	dbSNP
rs184402083	554	dbSNP
rs759793687	570	dbSNP
rs955484309	583	dbSNP
rs1161367767	584	dbSNP
rs1444272536	599	dbSNP
rs1414741664	610	dbSNP
rs1177885689	615	dbSNP
rs776769844	625	dbSNP
rs770822966	626	dbSNP
rs1417609195	629	dbSNP
rs1156673673	639	dbSNP
rs34394281	639	dbSNP
rs962937806	641	dbSNP
rs960412421	642	dbSNP
rs748709200	645	dbSNP
rs1164056548	659	dbSNP
rs781387051	663	dbSNP
rs1348069951	668	dbSNP
rs1443687714	670	dbSNP
rs1033421666	682	dbSNP
rs1242112390	683	dbSNP
rs1407674589	688	dbSNP
rs1295494936	689	dbSNP
rs1356423525	692	dbSNP
rs1217114563	704	dbSNP
rs1016965412	705	dbSNP
rs903502093	707	dbSNP
rs1290046818	720	dbSNP
rs755136930	734	dbSNP
rs1043677159	741	dbSNP
rs1009868684	757	dbSNP
rs1001914804	760	dbSNP
rs1245645158	773	dbSNP
rs1258505856	776	dbSNP
rs970187881	777	dbSNP
rs1024773589	780	dbSNP
rs201659549	781	dbSNP
rs1014051889	790	dbSNP
rs1229605265	794	dbSNP
rs1359243143	795	dbSNP
rs1353484818	811	dbSNP
rs757390303	827	dbSNP
rs944132117	830	dbSNP
rs1400543255	831	dbSNP
rs1414518778	835	dbSNP
rs1282844410	840	dbSNP
rs1364683237	848	dbSNP
rs780151730	849	dbSNP
rs1213144864	851	dbSNP
rs181782850	853	dbSNP
rs1485488490	854	dbSNP
rs1423741441	859	dbSNP
rs912678942	863	dbSNP
rs1476348600	869	dbSNP
rs1000090245	873	dbSNP
rs1418725624	874	dbSNP
rs1049796125	875	dbSNP
rs1350725836	879	dbSNP
rs1375654376	881	dbSNP
rs932676523	887	dbSNP
rs1332607230	904	dbSNP
rs1347254122	910	dbSNP
rs1431953359	920	dbSNP
rs1167169344	923	dbSNP
rs760578858	930	dbSNP
rs1236545087	944	dbSNP
rs974372482	946	dbSNP
rs1347406734	947	dbSNP
rs1217756502	958	dbSNP
rs1038710194	961	dbSNP
rs943005244	971	dbSNP
rs13194	978	dbSNP
rs7052295	983	dbSNP
rs1267321379	985	dbSNP
rs1216262159	995	dbSNP
rs1222272430	1018	dbSNP
rs1247310474	1023	dbSNP
rs908719864	1024	dbSNP
rs1194943861	1031	dbSNP
rs1051547534	1037	dbSNP
rs1453276079	1052	dbSNP
rs763936198	1061	dbSNP
rs1409740783	1073	dbSNP
rs1401614074	1080	dbSNP
rs1490605541	1082	dbSNP
rs960863977	1089	dbSNP
rs1355036342	1094	dbSNP
rs760584096	1096	dbSNP
rs929038845	1105	dbSNP
rs55822441	1111	dbSNP
rs766975380	1117	dbSNP
rs972883742	1124	dbSNP
rs1305337814	1128	dbSNP
rs962970685	1140	dbSNP
rs1434417428	1143	dbSNP
rs772149832	1162	dbSNP
rs1342346100	1165	dbSNP
rs1281267360	1166	dbSNP
rs1404177826	1167	dbSNP
rs75970188	1174	dbSNP
rs967718861	1178	dbSNP
rs758993128	1185	dbSNP
rs189325652	1186	dbSNP
rs1009489175	1187	dbSNP
rs970430783	1200	dbSNP
rs1450492080	1201	dbSNP
rs892341611	1202	dbSNP
rs1049740	1203	dbSNP
rs372914288	1209	dbSNP
rs1234222545	1215	dbSNP
rs747547446	1221	dbSNP
rs1158867832	1227	dbSNP
rs184308029	1247	dbSNP
rs1460661980	1251	dbSNP
rs1014329843	1264	dbSNP
rs1463348288	1270	dbSNP
rs1166211733	1272	dbSNP
rs1394423094	1277	dbSNP
rs1403635718	1281	dbSNP
rs4351535	1288	dbSNP
rs111358152	1290	dbSNP
rs1008721723	1295	dbSNP
rs1171501462	1296	dbSNP
rs1467225637	1299	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.