>TRAM1|NM_014294|3'UTR
   1 TGAATTATAAACTAATTGATTAATGTCCCCAAAGAAATCTGCTTTCTACTATATCTTTCAGCATTAGAGATTTTTCTGTT
  81 CTTGAAAATACAGTCTGTGCTCTTTGATTTTTGCTATTGTACGGTTTCATGCATTTTTTTAAAGGGCATTTGAGGGGAGG
 161 ATTATTGCTATGAATGAAAAAAATATTTTAGCTTAGACTAAGCTACCTGCCTTCAAAATAGTTTAGGGACCACCACCATA
 241 TTTTATTTTGTTTTTATTTTTGAACATTTTTCTAATGATTTGGAGAGAAAACTATTTACAAAAATTCCACATATCAGTGA
 321 TACAATTTCTTGCTGTCACCAATTTTTTATAATAGCAGAGTGGCCTGTTCTAAGAAGGCCATATTTTTTAAGTTATCTTT
 401 CAGGGTAACATGGAAATACTATAAAGTTGGATGTCAAACTTTAATATGTTTTCAGTGTTCTCTAATTTTTTGGAATTTTT
 481 GTAGACTTTACACCTGGAAAAAAAGATTTGTAAAATCACCGGAACAATTGTGTGCTTTATTTTATAGGTAGTGGTTATTA
 561 GTATTACATCCCCATTTTAAAAACAAAAACATAATAATCGTTACAACACGTGGAGTTTTACTAACATACATATTAAATCA
 641 AAGTATATTCTTAAAAGTACTTGTGAAGTAAAATCTTTCTTGTGCATTTTCAATACTTGTAAACTGGAAATCAGAAAATA
 721 TTTACTATGAACAGGAAAATCTGACATATAGCCCTTTTTGATATGTTTATTAATAATGATTCTTAATGGGGCTCATAATA
 801 AGTTTAATATGCACAGCATCTTAGAAAAGTTTAACCTGCAAACACTTTTAAAACATAATGCCTACTTGATTTATATCTAT
 881 AAAAAGACTGACAGGTAATTATATTTGGAAAACATTTAATGCACTAACTTTAAAGAAATTGAAAATTCAGGTGGATAAAT
 961 AGTCTTACAAAAGACAATGTGCTTTATGTTATACCTATAGCTTTGGTCCCATCTTTAATTGAGAAACATTTATCTGTATA
1041 AAACATATTTTTGGATAAATATATATATATATATTTGTATCTCTACAGAAAGGCTCTAAAAAGCATTTGAGGAAAATATT
1121 TGGTTCCCTTTTCTATAATCATCCTTTAAGATTCTTATAGCTACATTTGGTTTATTCATCATATTTACAGTATATATATT
1201 GTTCTTTTCAGTGTTCACATCTTGTTCCCCATTTCTCACTTGTGTCACCAGCTGTTTGTGCCATTTTTAGTGTAAAAGTT
1281 GCAGACCTATTAGATCTGCAGTTTAAGTTGCCATGCTGCTAGGAAATTGTCCTTTTTCTTTCTAGCTGTTAACCTACTTC
1361 CTGGAAAAAGTAGTAGCTCTCTGTAGCATTATGGAGTTTCAGTGGAACCAAATTTTTGCCATTAAAAACTGGCATTATAC
1441 TGAACTATACATTGAGAAATCAATCAAAATAAAAATTTTTACTTTCACAAGTTGTATCCTGGATGTTTCTGTCATTGTTG
1521 GTGTTTAGGCTATTTTGGTATATAACCTCATTAAAATGTACCATATTTAAAACACTTCATAGACATTCAGAATAACCCTT
1601 TTCAAAATTGTGTTCTGCAAATAAACAGATTTGTTCCACAGAATATTGGTGAGGACTACTGGGGGGATAGATGGTCATAA
1681 CCAAATAAATCTGGGAATCACTGGATTAAACTAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' guGAGUCGC-----CGGCA-AAGGGGUu 5' :||||:| :| | ||||||| Target 5' ttTTCAGTGTTCACATCTTGTTCCCCAt 3'	1205 - 1232	148.00	-17.10
2	miRNA 3' gugagucgccggcaaAGGGGUu 5' |||||| Target 5' actaattgattaatgTCCCCAa 3'	11 - 32	120.00	-12.20
3	miRNA 3' gugagucgccggcaaAGGGGUu 5' |||||| Target 5' gttattagtattacaTCCCCAt 3'	554 - 575	120.00	-10.32

Mutant ID	Mutant Position	Mutant Source
COSN30163947	2	COSMIC
COSN31525410	34	COSMIC
COSN31571219	55	COSMIC
COSN31553967	69	COSMIC
COSN31599834	109	COSMIC
COSN20549688	122	COSMIC
COSN28862050	134	COSMIC
COSN30169055	161	COSMIC
COSN30172148	314	COSMIC
COSN31545638	319	COSMIC
COSN31527806	329	COSMIC
COSN31606896	533	COSMIC
COSN31565044	536	COSMIC
COSN17556289	554	COSMIC
COSM5650800	658	COSMIC
COSM8855594	675	COSMIC
COSN31532950	752	COSMIC
COSN28888863	767	COSMIC
COSN24096556	875	COSMIC
COSN28164882	902	COSMIC
COSN30159364	927	COSMIC
COSN31601327	958	COSMIC
COSN26679720	968	COSMIC
COSN29721370	1059	COSMIC
COSN20446622	1082	COSMIC
COSN31973168	1288	COSMIC
COSN31578660	1316	COSMIC
COSN22621076	1337	COSMIC
COSN31591754	1346	COSMIC
COSN31530891	1372	COSMIC
COSN31484882	1384	COSMIC
COSN26556332	1433	COSMIC
COSN31549128	1467	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1390514578	4	dbSNP
rs1011905375	8	dbSNP
rs1426311018	15	dbSNP
rs1269718417	18	dbSNP
rs775631270	20	dbSNP
rs1463420957	22	dbSNP
rs1258254436	30	dbSNP
rs767837236	34	dbSNP
rs1482634666	35	dbSNP
rs1277631831	37	dbSNP
rs762649393	43	dbSNP
rs1309898786	48	dbSNP
rs773098002	51	dbSNP
rs1003931257	53	dbSNP
rs1166569812	57	dbSNP
rs886776685	61	dbSNP
rs1415026936	62	dbSNP
rs1048087627	66	dbSNP
rs1160944648	66	dbSNP
rs1399391134	67	dbSNP
rs1318215498	76	dbSNP
rs1360303091	76	dbSNP
rs995530595	80	dbSNP
rs1026654401	91	dbSNP
rs1373957619	99	dbSNP
rs1229227148	100	dbSNP
rs995740638	102	dbSNP
rs1290514884	106	dbSNP
rs548985553	113	dbSNP
rs1037199887	122	dbSNP
rs116362993	123	dbSNP
rs1057151552	129	dbSNP
rs1207944516	131	dbSNP
rs934745441	141	dbSNP
rs924651527	146	dbSNP
rs978774108	149	dbSNP
rs1482550856	152	dbSNP
rs891200001	155	dbSNP
rs921210677	160	dbSNP
rs559703754	170	dbSNP
rs1272002815	171	dbSNP
rs1197137023	176	dbSNP
rs539984552	177	dbSNP
rs1339853970	185	dbSNP
rs1273964034	186	dbSNP
rs1230978100	187	dbSNP
rs1311651069	200	dbSNP
rs1374917387	201	dbSNP
rs1049773392	205	dbSNP
rs1347320375	211	dbSNP
rs1410557153	215	dbSNP
rs932758511	218	dbSNP
rs1285343403	222	dbSNP
rs577271031	224	dbSNP
rs1370233292	236	dbSNP
rs536108496	238	dbSNP
rs1238641297	241	dbSNP
rs990561340	245	dbSNP
rs1346877477	250	dbSNP
rs1327142334	252	dbSNP
rs1411821055	256	dbSNP
rs1279604817	263	dbSNP
rs1483081363	274	dbSNP
rs936591989	276	dbSNP
rs1249017561	278	dbSNP
rs1436794003	283	dbSNP
rs1199141750	285	dbSNP
rs1483220915	289	dbSNP
rs975707980	294	dbSNP
rs1397673933	299	dbSNP
rs1479245315	302	dbSNP
rs923892696	309	dbSNP
rs977932686	311	dbSNP
rs970577291	316	dbSNP
rs1468753588	317	dbSNP
rs1024826664	320	dbSNP
rs995163342	323	dbSNP
rs1471995710	332	dbSNP
rs768381695	333	dbSNP
rs1342730278	335	dbSNP
rs959120444	338	dbSNP
rs1271782131	349	dbSNP
rs1013550168	352	dbSNP
rs1183549568	361	dbSNP
rs1026644251	363	dbSNP
rs1437979910	363	dbSNP
rs1277734040	367	dbSNP
rs116057921	375	dbSNP
rs145354280	376	dbSNP
rs386726546	377	dbSNP
rs1016291457	384	dbSNP
rs879208624	385	dbSNP
rs1450565101	390	dbSNP
rs535198252	394	dbSNP
rs1008493957	395	dbSNP
rs1371394320	398	dbSNP
rs1227670221	403	dbSNP
rs1164018970	412	dbSNP
rs924683038	421	dbSNP
rs575914129	433	dbSNP
rs1349974624	434	dbSNP
rs1043512100	443	dbSNP
rs1365674079	447	dbSNP
rs1437167843	453	dbSNP
rs947424077	457	dbSNP
rs1444647392	458	dbSNP
rs190381878	464	dbSNP
rs1244942902	466	dbSNP
rs1266719952	472	dbSNP
rs746933192	472	dbSNP
rs775634496	476	dbSNP
rs771121478	480	dbSNP
rs781712922	481	dbSNP
rs1272883625	488	dbSNP
rs1336562769	489	dbSNP
rs1333976887	491	dbSNP
rs1468536220	493	dbSNP
rs975359214	497	dbSNP
rs1427975888	500	dbSNP
rs1177811731	504	dbSNP
rs1457118913	507	dbSNP
rs1188279213	508	dbSNP
rs1370307203	510	dbSNP
rs1049884789	511	dbSNP
rs555811124	520	dbSNP
rs115513409	521	dbSNP
rs1164550075	532	dbSNP
rs1389090808	541	dbSNP
rs1054457470	547	dbSNP
rs749390977	553	dbSNP
rs1287022635	562	dbSNP
rs1384097027	566	dbSNP
rs923868025	568	dbSNP
rs912433992	571	dbSNP
rs1042668269	578	dbSNP
rs949299453	581	dbSNP
rs1315101721	584	dbSNP
rs1396881001	589	dbSNP
rs769598306	590	dbSNP
rs951133286	591	dbSNP
rs917723990	592	dbSNP
rs1218444919	594	dbSNP
rs748051024	594	dbSNP
rs1378492430	597	dbSNP
rs1026752310	599	dbSNP
rs268566	599	dbSNP
rs1187452234	600	dbSNP
rs1413034041	600	dbSNP
rs969086717	601	dbSNP
rs764205144	609	dbSNP
rs553401846	610	dbSNP
rs1460275861	612	dbSNP
rs1013203587	613	dbSNP
rs973752761	617	dbSNP
rs1165008527	619	dbSNP
rs896059900	620	dbSNP
rs1308241157	621	dbSNP
rs1035846336	624	dbSNP
rs1351900774	625	dbSNP
rs1254719691	632	dbSNP
rs1183744522	633	dbSNP
rs1305229211	633	dbSNP
rs1357489787	636	dbSNP
rs746536106	636	dbSNP
rs1234593015	640	dbSNP
rs1253169904	643	dbSNP
rs1283131657	643	dbSNP
rs1195792863	647	dbSNP
rs1337832368	652	dbSNP
rs1210187575	654	dbSNP
rs1178256558	663	dbSNP
rs779563706	663	dbSNP
rs961306248	669	dbSNP
rs539944885	671	dbSNP
rs1008859320	676	dbSNP
rs1175713897	679	dbSNP
rs1334469844	680	dbSNP
rs1043155264	682	dbSNP
rs750580987	682	dbSNP
rs955627616	682	dbSNP
rs763159826	686	dbSNP
rs1407113955	691	dbSNP
rs1291457600	692	dbSNP
rs997008593	697	dbSNP
rs893203626	700	dbSNP
rs1230311652	705	dbSNP
rs746190265	706	dbSNP
rs1307325964	708	dbSNP
rs1434055124	710	dbSNP
rs947419694	714	dbSNP
rs1389649856	721	dbSNP
rs899870217	722	dbSNP
rs1392576548	732	dbSNP
rs1293923391	734	dbSNP
rs1040101850	738	dbSNP
rs1297314324	745	dbSNP
rs1373008480	749	dbSNP
rs760089208	751	dbSNP
rs943964158	751	dbSNP
rs1441286572	756	dbSNP
rs779284391	764	dbSNP
rs773836294	765	dbSNP
rs1472630167	770	dbSNP
rs1410657955	773	dbSNP
rs757591230	775	dbSNP
rs772436380	778	dbSNP
rs929806762	778	dbSNP
rs756381754	779	dbSNP
rs1202689726	781	dbSNP
rs1251010357	781	dbSNP
rs761833254	785	dbSNP
rs754271953	788	dbSNP
rs764597233	790	dbSNP
rs1195215271	798	dbSNP
rs1319778622	802	dbSNP
rs1453656835	802	dbSNP
rs973850595	805	dbSNP
rs969537925	806	dbSNP
rs1023330911	807	dbSNP
rs1169280841	810	dbSNP
rs1350056199	817	dbSNP
rs991769023	823	dbSNP
rs903159428	824	dbSNP
rs1328262902	825	dbSNP
rs570920345	829	dbSNP
rs1440800947	838	dbSNP
rs1161311555	845	dbSNP
rs1364133926	845	dbSNP
rs769628459	848	dbSNP
rs1212935435	850	dbSNP
rs1272407147	851	dbSNP
rs1035917929	855	dbSNP
rs1045739416	859	dbSNP
rs1270978196	860	dbSNP
rs949275283	862	dbSNP
rs1467915864	865	dbSNP
rs999029933	869	dbSNP
rs903364479	870	dbSNP
rs1022208823	873	dbSNP
rs1474396519	874	dbSNP
rs1185388158	878	dbSNP
rs752851084	890	dbSNP
rs1162420994	893	dbSNP
rs1475949942	893	dbSNP
rs1392731811	895	dbSNP
rs1011695221	900	dbSNP
rs899902600	901	dbSNP
rs1039729924	913	dbSNP
rs544274489	919	dbSNP
rs944037813	928	dbSNP
rs1380003774	929	dbSNP
rs1484880001	930	dbSNP
rs1243743847	931	dbSNP
rs1216359839	932	dbSNP
rs891101059	935	dbSNP
rs1228724600	938	dbSNP
rs1286119569	947	dbSNP
rs1445141603	960	dbSNP
rs1447676658	962	dbSNP
rs185442564	968	dbSNP
rs1486404905	974	dbSNP
rs537496508	977	dbSNP
rs1185307445	978	dbSNP
rs1416781454	978	dbSNP
rs1472727631	982	dbSNP
rs929837643	986	dbSNP
rs1054948004	994	dbSNP
rs1410629528	995	dbSNP
rs1399407836	997	dbSNP
rs1318314947	998	dbSNP
rs1376144858	1006	dbSNP
rs200252262	1026	dbSNP
rs369296356	1027	dbSNP
rs1038600336	1033	dbSNP
rs1229246049	1034	dbSNP
rs1281012282	1036	dbSNP
rs1348345805	1038	dbSNP
rs937857682	1041	dbSNP
rs947856150	1045	dbSNP
rs564319137	1049	dbSNP
rs1208038843	1052	dbSNP
rs1483141951	1053	dbSNP
rs991841942	1053	dbSNP
rs1291181385	1058	dbSNP
rs1183556427	1060	dbSNP
rs1234951281	1060	dbSNP
rs1468989105	1060	dbSNP
rs919784994	1062	dbSNP
rs1432051668	1068	dbSNP
rs1214573305	1075	dbSNP
rs35679510	1075	dbSNP
rs753307668	1075	dbSNP
rs1374952942	1077	dbSNP
rs760358809	1079	dbSNP
rs1392159870	1080	dbSNP
rs1043024	1081	dbSNP
rs8993	1082	dbSNP
rs1275235104	1084	dbSNP
rs761669515	1093	dbSNP
rs34153988	1103	dbSNP
rs1373008020	1111	dbSNP
rs977683186	1115	dbSNP
rs189947286	1116	dbSNP
rs1342313065	1123	dbSNP
rs752051006	1124	dbSNP
rs986461080	1129	dbSNP
rs1197145616	1133	dbSNP
rs955617831	1135	dbSNP
rs115478532	1139	dbSNP
rs996956295	1158	dbSNP
rs768034096	1163	dbSNP
rs183876941	1165	dbSNP
rs1394527940	1166	dbSNP
rs1477616595	1183	dbSNP
rs1268349426	1190	dbSNP
rs999305937	1190	dbSNP
rs1394529796	1191	dbSNP
rs191341013	1192	dbSNP
rs1326287749	1201	dbSNP
rs1490915284	1204	dbSNP
rs1372937860	1227	dbSNP
rs1438594351	1238	dbSNP
rs1292742014	1239	dbSNP
rs532879390	1242	dbSNP
rs1326499056	1244	dbSNP
rs1229045718	1247	dbSNP
rs1204047748	1260	dbSNP
rs748877216	1270	dbSNP
rs1338302378	1271	dbSNP
rs1298114347	1274	dbSNP
rs1024724680	1275	dbSNP
rs1210089082	1283	dbSNP
rs1274177960	1286	dbSNP
rs1018382398	1289	dbSNP
rs1014288720	1295	dbSNP
rs1252988258	1296	dbSNP
rs896391777	1297	dbSNP
rs891132503	1306	dbSNP
rs1055021338	1312	dbSNP
rs1280877505	1313	dbSNP
rs779541291	1318	dbSNP
rs1345549985	1327	dbSNP
rs937931142	1330	dbSNP
rs898285997	1331	dbSNP
rs1038216241	1336	dbSNP
rs1459737239	1338	dbSNP
rs1325118706	1345	dbSNP
rs1385502557	1349	dbSNP
rs796685755	1351	dbSNP
rs1307051267	1355	dbSNP
rs1315554038	1356	dbSNP
rs1325399788	1359	dbSNP
rs994115937	1366	dbSNP
rs1294820491	1377	dbSNP
rs1337392099	1383	dbSNP
rs117154949	1408	dbSNP
rs1271851157	1432	dbSNP
rs908323383	1433	dbSNP
rs1486456280	1446	dbSNP
rs1207431784	1447	dbSNP
rs1259716016	1463	dbSNP
rs1356064016	1468	dbSNP
rs1170868495	1476	dbSNP
rs986410177	1477	dbSNP
rs1038268236	1494	dbSNP
rs947896635	1497	dbSNP
rs749963168	1498	dbSNP
rs1474354335	1502	dbSNP
rs1163898750	1517	dbSNP
rs1364190003	1523	dbSNP
rs166537	1524	dbSNP
rs1269643244	1525	dbSNP
rs1287087786	1527	dbSNP
rs1383615059	1527	dbSNP
rs1387649170	1533	dbSNP
rs1176855777	1536	dbSNP
rs975613364	1538	dbSNP
rs531043044	1542	dbSNP
rs761502419	1547	dbSNP
rs1205649116	1549	dbSNP
rs1312394518	1556	dbSNP
rs142221386	1564	dbSNP
rs977714201	1577	dbSNP
rs147646477	1582	dbSNP
rs1353056524	1588	dbSNP
rs1211611294	1589	dbSNP
rs967849515	1590	dbSNP
rs1484787987	1608	dbSNP
rs1345609607	1617	dbSNP
rs1250937705	1618	dbSNP
rs1420725760	1619	dbSNP
rs1156824886	1620	dbSNP
rs914825546	1626	dbSNP
rs1305590736	1627	dbSNP
rs60223808	1628	dbSNP
rs1389380581	1645	dbSNP
rs1174928753	1646	dbSNP
rs142596664	1649	dbSNP
rs1433689249	1653	dbSNP
rs1374501888	1654	dbSNP
rs1298582202	1658	dbSNP
rs553415911	1660	dbSNP
rs186489422	1661	dbSNP
rs1008352846	1665	dbSNP
rs79100715	1666	dbSNP
rs1282542717	1667	dbSNP
rs1345872331	1668	dbSNP
rs148464485	1675	dbSNP
rs1209679219	1677	dbSNP
rs78576848	1685	dbSNP
rs1481750819	1687	dbSNP
rs1025676064	1693	dbSNP
rs994596512	1697	dbSNP
rs1454622325	1701	dbSNP
rs1470205784	1704	dbSNP
rs898469398	1708	dbSNP
rs898444885	1711	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.