ID	Duplex structure	Position	Score	MFE
1	miRNA 3' uggucAACGGAGAU-GUCAGAAAu 5' | ||| | |:|||||| Target 5' cacccTGGCCAAAAGCGGTCTTTa 3'	837 - 860	134.00	-15.80
2	miRNA 3' ugGUCAACG-GAGAUGUCAGAaau 5' ::|| | ||||||||||| Target 5' acTGGTGACACTCTACAGTCTgac 3'	396 - 419	133.00	-21.52
3	miRNA 3' ugGUCAACGGAGAUG-UCAGAaau 5' | |||| ||||| ||||| Target 5' gcCTGTTG--TCTACAAGTCTaga 3'	118 - 139	129.00	-17.42

Mutant ID	Mutant Position	Mutant Source
COSN31592348	4	COSMIC
COSN30513746	7	COSMIC
COSN30134401	8	COSMIC
COSN30523855	14	COSMIC
COSN30515449	25	COSMIC
COSN26980342	28	COSMIC
COSN31492781	66	COSMIC
COSN31541933	86	COSMIC
COSN31579413	91	COSMIC
COSN31501347	99	COSMIC
COSN30130118	282	COSMIC
COSN15711161	297	COSMIC
COSN29287312	574	COSMIC
COSN5521200	854	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs756978063	7	dbSNP
rs770170481	8	dbSNP
rs577608430	9	dbSNP
rs951777550	12	dbSNP
rs372326423	14	dbSNP
rs766728413	15	dbSNP
rs752120035	16	dbSNP
rs760126578	17	dbSNP
rs200520220	25	dbSNP
rs375713236	27	dbSNP
rs756954500	28	dbSNP
rs778755925	37	dbSNP
rs1288245610	39	dbSNP
rs1347970970	42	dbSNP
rs953217015	46	dbSNP
rs750253432	48	dbSNP
rs753428026	49	dbSNP
rs758221188	51	dbSNP
rs1277411830	54	dbSNP
rs943202830	56	dbSNP
rs911798729	61	dbSNP
rs1040716412	63	dbSNP
rs1243105211	64	dbSNP
rs528917858	68	dbSNP
rs1321606401	70	dbSNP
rs1294487873	80	dbSNP
rs1416090013	85	dbSNP
rs548784518	86	dbSNP
rs1221804965	87	dbSNP
rs112952087	91	dbSNP
rs1382393185	92	dbSNP
rs1386726043	93	dbSNP
rs920220004	99	dbSNP
rs930387446	105	dbSNP
rs1457262261	108	dbSNP
rs1055523402	110	dbSNP
rs1167098943	119	dbSNP
rs1476802884	124	dbSNP
rs1185031719	129	dbSNP
rs1261046804	129	dbSNP
rs1260700378	131	dbSNP
rs916752526	133	dbSNP
rs757409553	138	dbSNP
rs1428916012	141	dbSNP
rs559876209	169	dbSNP
rs1383303522	182	dbSNP
rs1468361605	184	dbSNP
rs1423978938	185	dbSNP
rs1274207779	187	dbSNP
rs1235622293	193	dbSNP
rs1042376554	203	dbSNP
rs1047121449	205	dbSNP
rs906783185	207	dbSNP
rs1327808662	214	dbSNP
rs1000941315	216	dbSNP
rs756537256	225	dbSNP
rs778974081	226	dbSNP
rs1287927087	230	dbSNP
rs895329656	231	dbSNP
rs1052397033	232	dbSNP
rs531509973	240	dbSNP
rs1422863968	247	dbSNP
rs893781376	258	dbSNP
rs577162754	264	dbSNP
rs146028748	267	dbSNP
rs573772521	268	dbSNP
rs1196947935	273	dbSNP
rs1300346335	283	dbSNP
rs1381200398	284	dbSNP
rs1245920865	290	dbSNP
rs1315773182	294	dbSNP
rs1215980321	295	dbSNP
rs1323587631	318	dbSNP
rs1456801655	318	dbSNP
rs1213842203	321	dbSNP
rs539476546	323	dbSNP
rs546957005	327	dbSNP
rs1260356101	330	dbSNP
rs1025536172	332	dbSNP
rs897419348	334	dbSNP
rs994382341	342	dbSNP
rs953361244	344	dbSNP
rs1443874373	355	dbSNP
rs749823911	356	dbSNP
rs1386485066	362	dbSNP
rs35490938	363	dbSNP
rs1019316366	366	dbSNP
rs566867541	369	dbSNP
rs971941055	382	dbSNP
rs769093832	389	dbSNP
rs920270463	393	dbSNP
rs951668571	396	dbSNP
rs1398659816	403	dbSNP
rs1170921198	406	dbSNP
rs868470986	411	dbSNP
rs1364014280	413	dbSNP
rs6774347	414	dbSNP
rs915657598	415	dbSNP
rs945336901	426	dbSNP
rs370267267	439	dbSNP
rs778967322	441	dbSNP
rs556000311	448	dbSNP
rs1404402058	449	dbSNP
rs1275831298	454	dbSNP
rs1209568254	456	dbSNP
rs1348112968	465	dbSNP
rs1282078889	472	dbSNP
rs746664306	485	dbSNP
rs1237666941	503	dbSNP
rs1372428786	505	dbSNP
rs748293061	519	dbSNP
rs1046561654	522	dbSNP
rs139997357	524	dbSNP
rs1190764005	533	dbSNP
rs1444240422	538	dbSNP
rs1338664469	543	dbSNP
rs936669078	549	dbSNP
rs935601697	551	dbSNP
rs1052614776	553	dbSNP
rs1055512803	554	dbSNP
rs1175833727	570	dbSNP
rs1418105443	572	dbSNP
rs1407640147	574	dbSNP
rs1337981038	575	dbSNP
rs553862515	579	dbSNP
rs1222141180	580	dbSNP
rs538434350	582	dbSNP
rs1259168508	591	dbSNP
rs1306441151	595	dbSNP
rs946704512	602	dbSNP
rs897324775	603	dbSNP
rs1230483055	611	dbSNP
rs1354144301	613	dbSNP
rs558216280	617	dbSNP
rs188136666	620	dbSNP
rs889200582	624	dbSNP
rs1356096994	625	dbSNP
rs1027227128	628	dbSNP
rs1009355302	634	dbSNP
rs1460707060	636	dbSNP
rs887465094	641	dbSNP
rs1005902249	651	dbSNP
rs35713895	651	dbSNP
rs1017657393	654	dbSNP
rs1018786410	657	dbSNP
rs964500052	661	dbSNP
rs1164907288	662	dbSNP
rs897633140	668	dbSNP
rs539885537	669	dbSNP
rs542883347	678	dbSNP
rs769662702	684	dbSNP
rs1485324404	688	dbSNP
rs1242392623	689	dbSNP
rs1217663667	691	dbSNP
rs1239116063	695	dbSNP
rs191766874	708	dbSNP
rs1272323590	720	dbSNP
rs1233763742	724	dbSNP
rs1176092217	726	dbSNP
rs1408103938	732	dbSNP
rs1295160514	735	dbSNP
rs991169073	736	dbSNP
rs1361500895	746	dbSNP
rs361236	747	dbSNP
rs1433316438	748	dbSNP
rs971179477	750	dbSNP
rs981572166	751	dbSNP
rs990844905	760	dbSNP
rs928156625	762	dbSNP
rs542283280	763	dbSNP
rs1427473512	767	dbSNP
rs375121481	771	dbSNP
rs1187840108	772	dbSNP
rs988383313	773	dbSNP
rs915483874	777	dbSNP
rs946745098	778	dbSNP
rs1252091870	780	dbSNP
rs75903332	787	dbSNP
rs1297629284	789	dbSNP
rs1469738304	793	dbSNP
rs889081228	802	dbSNP
rs1202506918	803	dbSNP
rs944822296	805	dbSNP
rs1296387225	807	dbSNP
rs1216486271	808	dbSNP
rs1369096150	812	dbSNP
rs879541262	820	dbSNP
rs108858	821	dbSNP
rs751526118	836	dbSNP
rs897685342	838	dbSNP
rs183925212	840	dbSNP
rs1048568387	841	dbSNP
rs1461925256	843	dbSNP
rs1027614104	853	dbSNP
rs375030816	854	dbSNP
rs1007518430	857	dbSNP
rs1013215877	860	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.

- 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.