>RNF121|NM_018320|3'UTR
   1 TGATGAAGAGCATCAGTGGAAAACCCACCCCACACGCCATGGACCTCAGGGCACTCTCCTCCCTGCCCACAAAGACCTCC
  81 TGGGTGGGAAAGACTCAAAGGGGTGCTTGGGCCACTCAGGACCCCTCTGGCTGTGTCGGACTGGGGAGGGATATGATGGA
 161 GAGCCAGCCAGTGGGGCTGTCAGCAGTGGGGGGCTTTTTAAAAGAAAACTATTTTGATGAATATATTTAAAAAACCTTTT
 241 TTTATTGTGGAGCATAGGAATTGCCCCCCTCCAGGCTTCACCCTCCCTGCCTAAGCAGGGTGGGGGCAGAGCCATGACAT
 321 TTTTGGTTTAAAGGAGCCTTCTCATCTCTGGCCGAGAACACTGCTGGGCTCCCAGGTAGCTGAAGGCCTCAGCCCACCCA
 401 CTCCCTTCTTCCCTGTGTGGGGCTCAAGCCTGGTGCACTTAGTATAGAAGAGCTAACGTACTCAGGCTTGTGCCACGGGT
 481 GAGCACTGAGGCCCCAGGTGTCCTCCCTCCCCCACTGTCTGGGACACAGGACAGGTGTTGCGTCTGACTCTGTCTCCCCC
 561 TGTCCAAGAGCTATAGGCTGGTTGAGGCAAGTGGAACCTTATCAGTGACTTGGCCAACAAGGGAAGCCTTGGGGCCTCAG
 641 AATTCATTGAAGATTTGTTTTGGGACAACTGGAAGAATCAGCACCATGGATACTCGCCCGTGGGCAGGGCTGTGGTCAGC
 721 CAAGAATAGAGGTCATATAGTTGGGCCATGGGGCAATTGTTGGCAGCCATGACTGACGGCCCCTTGTGCAAGCTCATTCT
 801 TCAGTGGATCCTTCAGTGGAGTGGTGACACGTGCCAGCGGTCACTCTGCCACATCACTTCCTTCTTGAAAGCCTAAGAGT
 881 GCGTATGCGTGTGTGTGTGTGTGTGTGTGTGTACGTGAGGCAGAAGAATATGACAGATGTCAGCTTCTCCCTGTGTCAGA
 961 TGATATGTCCTTGAAGCTGGCAGGGGATTCCCAGTCCAGGGAATATCCCTACATGATTTTTGTCATTCATGGGTCCTCCC
1041 TGGGCCTGACAACGGGAGTGGTGGGTGGGTGAGAACAGGTTCTGAGAGGGGGCTTCTAGGAAGGCCCCAGCCCTAGTCCT
1121 AGGCGACTTGAATAAAGTGGGAGGTCAAACATGGGCCTTCGGCATTGGGTTTCTAGGGAATATGGCTCAGTAGGGCCTGC
1201 CCCTTCAAGGGCAGCAAGAACGAAGCAGGATGTTGTCACTCCCGTCTGAATAAAGCTCCATGAGCTGGGTTGGAAAGCTG
1281 AAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gcggCAGGGCCCUCG---GGGUGAGu 5' | |:| ||| ||||||| Target 5' gaagGCCTC---AGCCCACCCACTCc 3'	382 - 404	144.00	-18.30
2	miRNA 3' gcGGCAGGGCCCUCGGGGUGAGu 5' |:| | |||| :|||| || Target 5' agCTGGCAGGGGATTCCCAGTCc 3'	975 - 997	133.00	-23.20
3	miRNA 3' gcgGCAGGGCCCUCGGGGUGAgu 5' :||||: ||||||| Target 5' aggTGTCCTCCCTCCCCCACTgt 3'	496 - 518	132.00	-20.80

Mutant ID	Mutant Position	Mutant Source
COSN8407292	15	COSMIC
COSN31510294	26	COSMIC
COSN30458817	27	COSMIC
COSN1080398	31	COSMIC
COSN26586013	66	COSMIC
COSN29468852	93	COSMIC
COSN30544519	93	COSMIC
COSN28632150	105	COSMIC
COSN26638947	156	COSMIC
COSN16167451	160	COSMIC
COSN30127144	169	COSMIC
COSN31564479	178	COSMIC
COSN30127081	183	COSMIC
COSN31583176	205	COSMIC
COSN31588824	244	COSMIC
COSN31589293	248	COSMIC
COSN21345214	437	COSMIC
COSN26498742	569	COSMIC
COSN31778938	908	COSMIC
COSN20108482	912	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1259204384	5	dbSNP
rs374984580	7	dbSNP
rs946843369	11	dbSNP
rs774861475	13	dbSNP
rs761499465	15	dbSNP
rs771783214	16	dbSNP
rs34947570	20	dbSNP
rs367671346	22	dbSNP
rs372451541	27	dbSNP
rs765859058	29	dbSNP
rs753759759	30	dbSNP
rs759533522	32	dbSNP
rs188341542	36	dbSNP
rs199822208	37	dbSNP
rs1349518079	39	dbSNP
rs180980375	40	dbSNP
rs750650377	44	dbSNP
rs566787622	65	dbSNP
rs1360393633	71	dbSNP
rs1055906223	83	dbSNP
rs1432171117	96	dbSNP
rs1355406994	101	dbSNP
rs386754841	104	dbSNP
rs533830383	104	dbSNP
rs1055452	105	dbSNP
rs185304890	106	dbSNP
rs1256830864	109	dbSNP
rs912649127	113	dbSNP
rs944078691	118	dbSNP
rs1417194128	121	dbSNP
rs3829215	129	dbSNP
rs1266555133	130	dbSNP
rs897683673	133	dbSNP
rs924132294	134	dbSNP
rs994653334	138	dbSNP
rs1201591286	139	dbSNP
rs1486397890	148	dbSNP
rs1258869039	154	dbSNP
rs1421584929	155	dbSNP
rs190408514	157	dbSNP
rs1275494090	158	dbSNP
rs1215553949	161	dbSNP
rs1412283766	163	dbSNP
rs1473345182	166	dbSNP
rs575930870	168	dbSNP
rs1292044608	169	dbSNP
rs1416696294	172	dbSNP
rs1356220227	178	dbSNP
rs953454934	180	dbSNP
rs1156685496	181	dbSNP
rs565880584	212	dbSNP
rs538908113	213	dbSNP
rs757558784	216	dbSNP
rs1330565496	225	dbSNP
rs947753534	230	dbSNP
rs1399955485	237	dbSNP
rs1475882074	244	dbSNP
rs1046448369	246	dbSNP
rs368646464	248	dbSNP
rs1479430379	262	dbSNP
rs34646174	264	dbSNP
rs772613481	265	dbSNP
rs369628902	266	dbSNP
rs914011844	281	dbSNP
rs968109251	284	dbSNP
rs1235729805	292	dbSNP
rs899029618	303	dbSNP
rs1286668231	319	dbSNP
rs1280415634	320	dbSNP
rs980041165	320	dbSNP
rs926806889	325	dbSNP
rs1211225962	335	dbSNP
rs995077991	340	dbSNP
rs1323778475	342	dbSNP
rs1462904981	345	dbSNP
rs1350429265	349	dbSNP
rs181124629	354	dbSNP
rs953385544	355	dbSNP
rs1482533880	370	dbSNP
rs1056686208	375	dbSNP
rs771978247	376	dbSNP
rs916929649	382	dbSNP
rs1454090990	383	dbSNP
rs565286944	395	dbSNP
rs532364485	399	dbSNP
rs371295709	401	dbSNP
rs1489057033	414	dbSNP
rs1406782366	416	dbSNP
rs1433594932	417	dbSNP
rs1035971181	422	dbSNP
rs1177909703	423	dbSNP
rs897608317	434	dbSNP
rs550680412	436	dbSNP
rs1049407026	438	dbSNP
rs888917241	440	dbSNP
rs867053062	443	dbSNP
rs568984392	448	dbSNP
rs137972494	458	dbSNP
rs11235416	459	dbSNP
rs556778298	477	dbSNP
rs566540209	478	dbSNP
rs1392887501	479	dbSNP
rs1296709762	483	dbSNP
rs1046078810	484	dbSNP
rs1372845036	487	dbSNP
rs113699392	498	dbSNP
rs979500237	499	dbSNP
rs1303809406	502	dbSNP
rs1034273010	507	dbSNP
rs959541247	509	dbSNP
rs992840915	511	dbSNP
rs898912968	515	dbSNP
rs186780651	516	dbSNP
rs1294746666	538	dbSNP
rs918194787	541	dbSNP
rs994690393	542	dbSNP
rs940263799	543	dbSNP
rs1343758996	548	dbSNP
rs1373228853	557	dbSNP
rs1202367618	560	dbSNP
rs1466843580	563	dbSNP
rs1372914472	566	dbSNP
rs539719796	569	dbSNP
rs1009255520	574	dbSNP
rs1199369722	583	dbSNP
rs1430771417	588	dbSNP
rs971738690	592	dbSNP
rs918921659	595	dbSNP
rs1018853716	604	dbSNP
rs1453560916	611	dbSNP
rs570442062	618	dbSNP
rs191733941	622	dbSNP
rs542364922	624	dbSNP
rs1224980070	628	dbSNP
rs1431760645	631	dbSNP
rs1541306	653	dbSNP
rs118006000	661	dbSNP
rs1232149668	672	dbSNP
rs1353061679	681	dbSNP
rs765435402	691	dbSNP
rs1314305862	693	dbSNP
rs543482255	696	dbSNP
rs777849190	697	dbSNP
rs901793045	700	dbSNP
rs1369309960	701	dbSNP
rs1164912268	705	dbSNP
rs1009483110	706	dbSNP
rs1474255148	708	dbSNP
rs183954725	715	dbSNP
rs1188371286	717	dbSNP
rs1437394163	724	dbSNP
rs773317536	730	dbSNP
rs1248705998	732	dbSNP
rs555189220	736	dbSNP
rs1193847893	757	dbSNP
rs1258602407	765	dbSNP
rs983363888	766	dbSNP
rs754042130	768	dbSNP
rs763162954	778	dbSNP
rs1434101080	787	dbSNP
rs1001373065	789	dbSNP
rs928218767	793	dbSNP
rs1033747522	796	dbSNP
rs1334745617	802	dbSNP
rs940397139	804	dbSNP
rs1299040808	822	dbSNP
rs1449459950	829	dbSNP
rs971673092	832	dbSNP
rs1225591943	839	dbSNP
rs76430787	840	dbSNP
rs779330877	846	dbSNP
rs115432361	847	dbSNP
rs565406832	850	dbSNP
rs1050167385	852	dbSNP
rs752131230	857	dbSNP
rs992370319	881	dbSNP
rs888968467	882	dbSNP
rs1238507884	883	dbSNP
rs1441288068	883	dbSNP
rs532395085	884	dbSNP
rs1467027360	886	dbSNP
rs1040348750	887	dbSNP
rs1223389826	888	dbSNP
rs10532049	889	dbSNP
rs1160389847	889	dbSNP
rs1209898030	889	dbSNP
rs1303049814	889	dbSNP
rs1324864284	889	dbSNP
rs1362260572	889	dbSNP
rs1382950706	889	dbSNP
rs3841467	889	dbSNP
rs398016602	889	dbSNP
rs774378740	889	dbSNP
rs774483930	889	dbSNP
rs1263309806	890	dbSNP
rs371425432	891	dbSNP
rs746773435	892	dbSNP
rs1192710461	896	dbSNP
rs1452407026	898	dbSNP
rs1248377644	902	dbSNP
rs961645845	904	dbSNP
rs1446381962	911	dbSNP
rs137970270	912	dbSNP
rs1491035249	913	dbSNP
rs1188945975	915	dbSNP
rs768533109	916	dbSNP
rs1279238878	919	dbSNP
rs999527833	926	dbSNP
rs918932999	937	dbSNP
rs1447537466	941	dbSNP
rs1430577985	943	dbSNP
rs1325229443	945	dbSNP
rs1300036384	948	dbSNP
rs1400701337	949	dbSNP
rs930302030	952	dbSNP
rs1375057959	963	dbSNP
rs1172078727	964	dbSNP
rs755332974	967	dbSNP
rs1172976383	970	dbSNP
rs1475004917	976	dbSNP
rs1030983386	978	dbSNP
rs910330930	984	dbSNP
rs1011437558	986	dbSNP
rs1256332001	987	dbSNP
rs1208349296	988	dbSNP
rs1392511989	994	dbSNP
rs780872273	1001	dbSNP
rs1461293099	1002	dbSNP
rs1023702082	1006	dbSNP
rs1367984923	1007	dbSNP
rs1219321142	1008	dbSNP
rs1354943672	1009	dbSNP
rs1429171899	1012	dbSNP
rs943101893	1014	dbSNP
rs1450419849	1015	dbSNP
rs1359970640	1017	dbSNP
rs187898945	1023	dbSNP
rs1409217318	1025	dbSNP
rs1040268907	1029	dbSNP
rs970000785	1033	dbSNP
rs562528192	1037	dbSNP
rs371605528	1039	dbSNP
rs982239457	1041	dbSNP
rs767933325	1044	dbSNP
rs1473535018	1051	dbSNP
rs1035244773	1053	dbSNP
rs190799345	1054	dbSNP
rs796082356	1055	dbSNP
rs1199878502	1063	dbSNP
rs1042301125	1074	dbSNP
rs1291444722	1085	dbSNP
rs903871989	1088	dbSNP
rs1230114117	1090	dbSNP
rs920282475	1092	dbSNP
rs1209154783	1093	dbSNP
rs116012482	1097	dbSNP
rs1441173941	1102	dbSNP
rs1463500100	1108	dbSNP
rs1397911450	1114	dbSNP
rs986321671	1115	dbSNP
rs560300157	1125	dbSNP
rs769624708	1126	dbSNP
rs1473187869	1129	dbSNP
rs944530361	1139	dbSNP
rs895268113	1144	dbSNP
rs903118717	1146	dbSNP
rs1474966283	1152	dbSNP
rs772772293	1153	dbSNP
rs1240361440	1157	dbSNP
rs934537097	1161	dbSNP
rs182344487	1162	dbSNP
rs770547802	1166	dbSNP
rs3814722	1168	dbSNP
rs1223861455	1171	dbSNP
rs774032448	1181	dbSNP
rs893822549	1188	dbSNP
rs1010905526	1196	dbSNP
rs1379439665	1200	dbSNP
rs1311092248	1207	dbSNP
rs1024175676	1223	dbSNP
rs961492755	1229	dbSNP
rs1360231044	1239	dbSNP
rs1449615406	1240	dbSNP
rs1290857770	1244	dbSNP
rs1354431963	1245	dbSNP
rs1350010716	1255	dbSNP
rs759214575	1257	dbSNP
rs1417436375	1258	dbSNP
rs1381794642	1270	dbSNP

- 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.