ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgAGACGAGUCUUGAUGUGAg 5' |:|| | | |||||||| Target 5' taTTTG-TAAATACTACACTa 3'	516 - 535	154.00	-11.60
2	miRNA 3' cgaGACGA-GU-CUUGAUGUGAg 5' |||:| || ||| ||||| Target 5' ttgCTGTTACACAAACAACACTa 3'	435 - 457	130.00	-11.40
3	miRNA 3' cgAGACGAGUCUUGAUGUGAg 5' | || ||| :||||:| Target 5' caTGTGAACAGTCTTACATTt 3'	86 - 106	123.00	-10.60

Mutant ID	Mutant Position	Mutant Source
COSN30458083	26	COSMIC
COSN1120473	27	COSMIC
COSN31573228	52	COSMIC
COSN31586453	68	COSMIC
COSN20106828	95	COSMIC
COSN30152014	108	COSMIC
COSN28846832	121	COSMIC
COSN30138198	203	COSMIC
COSN16323588	210	COSMIC
COSN31562435	217	COSMIC
COSN26925496	318	COSMIC
COSN20106830	382	COSMIC
COSN23755531	421	COSMIC
COSN30212550	465	COSMIC
COSN5878418	493	COSMIC
COSN5878419	580	COSMIC
COSN21678883	647	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs772529151	2	dbSNP
rs773732502	4	dbSNP
rs1268528729	5	dbSNP
rs1321367754	7	dbSNP
rs760680316	10	dbSNP
rs551904436	16	dbSNP
rs776702262	17	dbSNP
rs759850747	20	dbSNP
rs764906769	21	dbSNP
rs767009889	26	dbSNP
rs752494232	29	dbSNP
rs1209847528	33	dbSNP
rs1262411274	35	dbSNP
rs1351375912	35	dbSNP
rs764236290	36	dbSNP
rs1364759290	37	dbSNP
rs781563976	38	dbSNP
rs1042753250	39	dbSNP
rs764062044	40	dbSNP
rs746191214	46	dbSNP
rs769380984	48	dbSNP
rs925596380	49	dbSNP
rs1315822069	50	dbSNP
rs751550916	50	dbSNP
rs756827035	53	dbSNP
rs1420815777	54	dbSNP
rs549179442	57	dbSNP
rs567380046	62	dbSNP
rs1312698028	72	dbSNP
rs1400976612	73	dbSNP
rs537958809	73	dbSNP
rs762903896	76	dbSNP
rs550298347	78	dbSNP
rs1039234466	84	dbSNP
rs900680534	89	dbSNP
rs997604577	92	dbSNP
rs139115917	93	dbSNP
rs79427500	93	dbSNP
rs374489641	96	dbSNP
rs1474370616	98	dbSNP
rs1358903981	104	dbSNP
rs1052363122	123	dbSNP
rs942071125	125	dbSNP
rs1447363527	127	dbSNP
rs183243244	133	dbSNP
rs1485475769	136	dbSNP
rs576772470	137	dbSNP
rs996716316	138	dbSNP
rs1332295873	142	dbSNP
rs189098323	142	dbSNP
rs1029991320	143	dbSNP
rs1005366322	148	dbSNP
rs74861203	149	dbSNP
rs899141876	162	dbSNP
rs1316613064	163	dbSNP
rs371421725	164	dbSNP
rs573197875	165	dbSNP
rs1020538294	167	dbSNP
rs1459931127	169	dbSNP
rs776053816	175	dbSNP
rs1417498057	178	dbSNP
rs1171662901	179	dbSNP
rs1222624779	184	dbSNP
rs1293431979	186	dbSNP
rs1427890581	189	dbSNP
rs1444971214	190	dbSNP
rs1253526644	192	dbSNP
rs1001455740	201	dbSNP
rs1034235100	202	dbSNP
rs1466752334	205	dbSNP
rs374720345	212	dbSNP
rs992713175	213	dbSNP
rs1340983832	217	dbSNP
rs533940953	219	dbSNP
rs1476664907	226	dbSNP
rs1020517891	250	dbSNP
rs992375343	254	dbSNP
rs1385169181	259	dbSNP
rs1445275531	262	dbSNP
rs1439260266	266	dbSNP
rs1393850681	278	dbSNP
rs376381017	279	dbSNP
rs1411900651	289	dbSNP
rs766857811	289	dbSNP
rs1378678213	290	dbSNP
rs1165994484	294	dbSNP
rs1400387207	295	dbSNP
rs1461288934	297	dbSNP
rs1195434192	306	dbSNP
rs1395382304	306	dbSNP
rs1452441634	310	dbSNP
rs1251870618	312	dbSNP
rs1181863926	314	dbSNP
rs967182727	317	dbSNP
rs978888187	318	dbSNP
rs1270427867	327	dbSNP
rs369379635	327	dbSNP
rs1317369448	328	dbSNP
rs1195656529	329	dbSNP
rs1349055651	336	dbSNP
rs1282843563	341	dbSNP
rs1232769214	350	dbSNP
rs1345629650	351	dbSNP
rs1370861052	354	dbSNP
rs925637154	366	dbSNP
rs373312949	367	dbSNP
rs1381972717	368	dbSNP
rs1444440235	369	dbSNP
rs977527036	370	dbSNP
rs1334265125	371	dbSNP
rs1288395104	373	dbSNP
rs35002405	379	dbSNP
rs397786460	379	dbSNP
rs1358808270	382	dbSNP
rs974971488	385	dbSNP
rs922229053	388	dbSNP
rs1394380561	393	dbSNP
rs933552175	393	dbSNP
rs1051969961	398	dbSNP
rs1039119537	405	dbSNP
rs886580685	409	dbSNP
rs573928386	410	dbSNP
rs932469023	414	dbSNP
rs1180665127	415	dbSNP
rs1485767377	418	dbSNP
rs1284531489	420	dbSNP
rs1260093656	425	dbSNP
rs1209143483	426	dbSNP
rs749550536	440	dbSNP
rs1202209929	445	dbSNP
rs1243023375	448	dbSNP
rs1348643955	455	dbSNP
rs193033794	463	dbSNP
rs1050913454	465	dbSNP
rs1357572835	466	dbSNP
rs899174481	468	dbSNP
rs1185465510	477	dbSNP
rs1318897514	481	dbSNP
rs1402316268	489	dbSNP
rs1009414827	497	dbSNP
rs1469807847	498	dbSNP
rs1475356267	502	dbSNP
rs1419546731	512	dbSNP
rs1186327504	513	dbSNP
rs1485548876	514	dbSNP
rs1283696880	515	dbSNP
rs144339710	515	dbSNP
rs1233953531	517	dbSNP
rs1034347901	520	dbSNP
rs148627492	531	dbSNP
rs1033842763	534	dbSNP
rs1378192256	545	dbSNP
rs1174055838	546	dbSNP
rs1432845234	547	dbSNP
rs1343331731	549	dbSNP
rs959184649	552	dbSNP
rs1405222142	553	dbSNP
rs1396312562	559	dbSNP
rs1453368589	559	dbSNP
rs779464717	560	dbSNP
rs1025120047	563	dbSNP
rs530849752	563	dbSNP
rs1333414960	564	dbSNP
rs1440839238	565	dbSNP
rs769086500	567	dbSNP
rs950876277	567	dbSNP
rs577541863	568	dbSNP
rs1352429057	570	dbSNP
rs1222046132	571	dbSNP
rs1262957569	574	dbSNP
rs1324884709	575	dbSNP
rs545008029	579	dbSNP
rs1206189003	580	dbSNP
rs560377958	582	dbSNP
rs1213329037	584	dbSNP
rs963371403	588	dbSNP
rs1437246002	590	dbSNP
rs1296075807	591	dbSNP
rs375957202	592	dbSNP
rs183876354	595	dbSNP
rs186719454	597	dbSNP
rs1441468015	601	dbSNP
rs1372564554	603	dbSNP
rs1323258776	621	dbSNP
rs1457126698	624	dbSNP
rs201605887	628	dbSNP
rs1173060483	638	dbSNP
rs933381013	639	dbSNP
rs1389865001	640	dbSNP
rs1455928431	644	dbSNP
rs1251252828	646	dbSNP
rs561145274	657	dbSNP
rs1481110849	658	dbSNP
rs879260105	664	dbSNP
rs1197007696	669	dbSNP
rs1305559400	682	dbSNP
rs1273584453	687	dbSNP
rs747064639	694	dbSNP
rs912368251	701	dbSNP
rs755657602	703	dbSNP
rs1385719446	708	dbSNP
rs1373693783	709	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.