>RPS24|NM_001142285|3'UTR
   1 TGTCACTGCCATGGCCGCCTTGCTGCATTTCTGAGGATGCTTCATCTCTCCACCTTCTTCTCCACTCAGCAGCCAGCAGG
  81 GCACTGTGGAAATCGGAGTCACATGAGCTGGCACCTCTGTTCAGAACCCTCCAGGGCTCCACATCTCTCTCACCCAAATG
 161 CCAAAGACCTCCCCACGCCCCCACAATCCCCCACGACCTGGCCACTGGCCTCCCACCACCTTCCAGCTCCAGCGGCTCCT
 241 ACCACATTTAAGGCTTTCCTTCCTAGTTTTAATTTTTCCTCGTCAGCAGTTGATTTTATTATTTTCTTGTTTATTGGTAT
 321 TTTCCCACTAGAAATGAAGCTGCGTGAAGTTAGAGATTTTTTTTTTTGGTCTGTGTTCCTAATTAGCTCATTGCTATACC
 401 CCTGGCGCCCAGAACAATGCCTTGGACACAGTACGCAGTAGACTAAATAAATACTTGTTGAATGACTGACTGACGGAATG
 481 ACGGCTGTGTGGGGAGTGGATTGGGTCGTGAGGCAGAGGCTGCGGTGGAAACTCAGGCAGGAGGTGATGGTGGTTCTTGG
 561 GGCTGCGGAATGCCAAGTTTAGAAGCTCTTCCTCTGCTGTGGCACATGAACCGGTCACTCGAGAAGGCTTTTAGATTTAC
 641 TTTGCCTAATCCCCTCTTAGTGCATGTGGGGAAACTGAGGTACACAAAAGGAATTCCCCACCAAGTTAGGGGCAGAACCT
 721 AGCCCCCTTGTCTCCCAGATGGATATCTTCTTTTTTTTTTGAGACGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGT
 801 GGTACCATCTTGGCTCACTGCAACCTCTGCTTCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTGTCTGCGAT
 881 TACAGGTGCACACAACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTGGTCAGGGTGACC
 961 TCAAACTCCTGACCTCATGATCCACCCAGCTCAGCCTCCCAACGTGCTGGGATTACAGGCATGAGCCACCGTGCCTGGCT
1041 GGACATCTTGTTATTAAAGCTTCTTCTCTCTTTGTAGGGGAGGGGGAGATGCCTCTGGTGGAGAAGACCAGTGTGGCAGT
1121 GACTGTGTCTGTTAGTGAACCTGGTGGCTGGTTGAGGGTCTGTCGTGGTGACTGAGGACACATACAAAGTGCTTTTCTCA
1201 GTGGTCACCTTGGTGTTGGTGAATAAGGGTCAGAAGATGGCTCCTGTCCTAGGGCACTGCCAGTCGGTTTGGAAGCTGAA
1281 ATGCCTGCTTAGCAGTTTGAGGAAACACAGACCTTGGAGGATCTTCTGGTTGCCTCTTCAAGAATTCATTCTATTCCCCT
1361 TCTGCTCCCCAAATTTGCTTTTCTTGGGGTGGGTCTTGGTTGGCCTAAGCCAAGAAAGTATGGCATCTACTCCTTCCATA
1441 GCAATAGCTCAGGAATAGGCAGTGACCCAGACCTGAACCAATCAGTGCATGGAATTACCCCTGGCCAAAGTGGTTGATTG
1521 AGGCTGGGTGCAAGCAGAGTTGTGAGAAGGCTCCCATTTGGTGGTTGGAGAGATCGCACTTGCTCCAGAGGTCATAATGT
1601 GCAGATCTGAGGCTTGGAACTGCTGCAGACATTTTGCTACCACAAGTGAAGCCACCCTGACGACACAGTTGACAATTTGG
1681 AGCAGGGCAGAGCTGAGAGAACAGCAGGGAAACAGCCAGAGTCTTGCTCAAGCCTCCCTGAAGTATCTATACCCCTGGAC
1761 TCTAGTTATGGGGGCTAATAAATGTTATATACTGTTTAAGGTAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgGA-CGUAGUACU--UUACGGGAgc 5' || ||: | || ||||||:| Target 5' ccCTGGCGCCCAGAACAATGCCTTgg 3'	400 - 425	123.00	-16.60
2	miRNA 3' cgGACGUAGUACUUUACGGGAGc 5' :| | |:: ||| ||||| Target 5' atTTACTTTGCCTAATCCCCTCt 3'	635 - 657	121.00	-10.00
3	miRNA 3' cgGACGUAGUACUUUACGGGAGc 5' :|| | |||||||||: | Target 5' gtTTGGAAGCTGAAATGCCTGCt 3'	1267 - 1289	121.00	-13.90

Mutant ID	Mutant Position	Mutant Source
1187759	101	ClinVar
COSN31587210	39	COSMIC
COSN17370154	95	COSMIC
COSN1122889	332	COSMIC
COSN17606784	408	COSMIC
COSN17182052	875	COSMIC
COSN23453092	1005	COSMIC
COSN27091365	1032	COSMIC
COSN8189869	1166	COSMIC
COSN19493751	1318	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs536067246	11	dbSNP
rs183560808	12	dbSNP
rs1461562425	13	dbSNP
rs769160910	17	dbSNP
rs765237160	18	dbSNP
rs1394569670	22	dbSNP
rs939483296	26	dbSNP
rs1354588689	38	dbSNP
rs1453945212	40	dbSNP
rs1385664506	61	dbSNP
rs566667298	62	dbSNP
rs1286545030	69	dbSNP
rs532561240	77	dbSNP
rs992187317	78	dbSNP
rs538598613	95	dbSNP
rs1423505183	96	dbSNP
rs1422410713	97	dbSNP
rs114149556	101	dbSNP
rs1163989263	101	dbSNP
rs1446330450	104	dbSNP
rs1031462188	106	dbSNP
rs142851690	116	dbSNP
rs537930998	129	dbSNP
rs925561105	130	dbSNP
rs763076568	135	dbSNP
rs1219055451	143	dbSNP
rs1489924638	146	dbSNP
rs1052652717	148	dbSNP
rs1307524226	154	dbSNP
rs1247207476	155	dbSNP
rs554751714	156	dbSNP
rs1406691537	165	dbSNP
rs1291773480	166	dbSNP
rs915650254	168	dbSNP
rs377427583	176	dbSNP
rs540845064	178	dbSNP
rs560812258	179	dbSNP
rs973069775	185	dbSNP
rs1214980791	187	dbSNP
rs899554259	188	dbSNP
rs995179101	190	dbSNP
rs1297221370	193	dbSNP
rs1026609954	195	dbSNP
rs188440005	196	dbSNP
rs1226955895	201	dbSNP
rs151068494	206	dbSNP
rs1169651311	207	dbSNP
rs562732070	210	dbSNP
rs1429108412	213	dbSNP
rs1420243838	216	dbSNP
rs1195938663	217	dbSNP
rs1490717713	220	dbSNP
rs1225355735	226	dbSNP
rs1245532230	231	dbSNP
rs1035553838	234	dbSNP
rs531644670	235	dbSNP
rs1421857405	237	dbSNP
rs1447890240	240	dbSNP
rs751662641	247	dbSNP
rs1204374145	254	dbSNP
rs916674466	264	dbSNP
rs1385927846	280	dbSNP
rs548257521	282	dbSNP
rs1162556840	283	dbSNP
rs561879004	287	dbSNP
rs1283492425	291	dbSNP
rs1406461844	302	dbSNP
rs140313250	311	dbSNP
rs1297406772	317	dbSNP
rs1462373005	318	dbSNP
rs969406125	336	dbSNP
rs529802136	337	dbSNP
rs925269207	345	dbSNP
rs1428727940	356	dbSNP
rs1177022006	357	dbSNP
rs1481219401	357	dbSNP
rs35734754	357	dbSNP
rs397828117	357	dbSNP
rs576615131	357	dbSNP
rs5786317	357	dbSNP
rs200785086	369	dbSNP
rs1362500716	371	dbSNP
rs1433902126	372	dbSNP
rs1315380892	374	dbSNP
rs1360953795	376	dbSNP
rs1226156963	379	dbSNP
rs750225211	382	dbSNP
rs546497651	384	dbSNP
rs566736490	385	dbSNP
rs1234889647	392	dbSNP
rs996897418	403	dbSNP
rs192833809	407	dbSNP
rs753015915	408	dbSNP
rs1391779589	415	dbSNP
rs1334567228	418	dbSNP
rs141004459	421	dbSNP
rs1244015821	427	dbSNP
rs1177257356	432	dbSNP
rs1467741870	435	dbSNP
rs552483529	436	dbSNP
rs1158720478	438	dbSNP
rs150188824	444	dbSNP
rs1240488898	447	dbSNP
rs995251050	448	dbSNP
rs778227693	454	dbSNP
rs1048092311	467	dbSNP
rs1243148106	471	dbSNP
rs886760564	473	dbSNP
rs139078073	475	dbSNP
rs1003861379	476	dbSNP
rs1355574358	478	dbSNP
rs370707915	483	dbSNP
rs1401225209	488	dbSNP
rs1161289396	492	dbSNP
rs1036031371	508	dbSNP
rs758150474	509	dbSNP
rs1382936303	517	dbSNP
rs1288506665	519	dbSNP
rs959916608	520	dbSNP
rs1162064743	521	dbSNP
rs749862750	524	dbSNP
rs555857874	525	dbSNP
rs920397595	528	dbSNP
rs184005974	537	dbSNP
rs541091857	548	dbSNP
rs559326016	560	dbSNP
rs979474722	567	dbSNP
rs80146678	568	dbSNP
rs1289276416	581	dbSNP
rs1245007766	585	dbSNP
rs1359951649	589	dbSNP
rs1283106992	590	dbSNP
rs956938656	593	dbSNP
rs944312907	598	dbSNP
rs376774408	608	dbSNP
rs1041170386	609	dbSNP
rs142271202	613	dbSNP
rs79628312	614	dbSNP
rs1362724248	621	dbSNP
rs146163875	622	dbSNP
rs1366622738	627	dbSNP
rs1165502575	653	dbSNP
rs975615917	658	dbSNP
rs999247293	661	dbSNP
rs576010402	666	dbSNP
rs921422212	667	dbSNP
rs1478572455	676	dbSNP
rs1266301701	682	dbSNP
rs931078255	684	dbSNP
rs376530939	685	dbSNP
rs1208526499	688	dbSNP
rs1291836427	692	dbSNP
rs1014193593	693	dbSNP
rs1022765546	706	dbSNP
rs1269629552	713	dbSNP
rs1342501895	721	dbSNP
rs188152089	724	dbSNP
rs534205505	725	dbSNP
rs1263933324	726	dbSNP
rs1325953470	727	dbSNP
rs994715049	735	dbSNP
rs1444354671	743	dbSNP
rs1027806207	746	dbSNP
rs1426681959	750	dbSNP
rs1466705686	750	dbSNP
rs1248171977	751	dbSNP
rs950522402	751	dbSNP
rs983500676	751	dbSNP
rs1236974847	752	dbSNP
rs1437112414	752	dbSNP
rs1470588459	756	dbSNP
rs746551338	761	dbSNP
rs768158540	766	dbSNP
rs939692587	767	dbSNP
rs1409722143	778	dbSNP
rs1472119301	783	dbSNP
rs1056762584	787	dbSNP
rs776466271	799	dbSNP
rs911573546	802	dbSNP
rs1412407230	811	dbSNP
rs1335590104	814	dbSNP
rs1332629085	816	dbSNP
rs1333160985	821	dbSNP
rs965564610	821	dbSNP
rs1175094376	826	dbSNP
rs1354587943	829	dbSNP
rs1443792467	831	dbSNP
rs895776879	836	dbSNP
rs976972657	838	dbSNP
rs1156495795	839	dbSNP
rs1012778782	844	dbSNP
rs1022783083	847	dbSNP
rs368869583	853	dbSNP
rs747936688	856	dbSNP
rs1241278520	859	dbSNP
rs932803386	860	dbSNP
rs1000008423	863	dbSNP
rs1443129925	871	dbSNP
rs372435023	878	dbSNP
rs546732599	879	dbSNP
rs376462651	881	dbSNP
rs1350396976	883	dbSNP
rs560079260	886	dbSNP
rs1310467500	887	dbSNP
rs1242867866	889	dbSNP
rs773275465	891	dbSNP
rs370982100	895	dbSNP
rs1347665931	900	dbSNP
rs1019621932	901	dbSNP
rs1315779742	914	dbSNP
rs1399809237	915	dbSNP
rs1380615405	924	dbSNP
rs532383532	927	dbSNP
rs895823204	930	dbSNP
rs1438439603	932	dbSNP
rs373650390	933	dbSNP
rs1483618483	943	dbSNP
rs921453437	944	dbSNP
rs931483761	949	dbSNP
rs1446644249	954	dbSNP
rs1287141600	960	dbSNP
rs1219276340	963	dbSNP
rs1482696344	966	dbSNP
rs984273021	968	dbSNP
rs545328381	970	dbSNP
rs1044329282	974	dbSNP
rs1431136932	975	dbSNP
rs552165146	991	dbSNP
rs1380688893	993	dbSNP
rs563433674	1001	dbSNP
rs774242599	1003	dbSNP
rs995059685	1005	dbSNP
rs1027506479	1011	dbSNP
rs1422484706	1012	dbSNP
rs1364806812	1017	dbSNP
rs191450498	1018	dbSNP
rs1165384911	1021	dbSNP
rs1057235655	1031	dbSNP
rs142817294	1032	dbSNP
rs1415967896	1033	dbSNP
rs948663403	1046	dbSNP
rs1184889609	1051	dbSNP
rs1477591460	1060	dbSNP
rs1264145876	1062	dbSNP
rs1004641715	1063	dbSNP
rs1305652533	1066	dbSNP
rs1044310589	1069	dbSNP
rs904420043	1075	dbSNP
rs974416079	1077	dbSNP
rs548211729	1079	dbSNP
rs568123369	1088	dbSNP
rs533643051	1090	dbSNP
rs767694787	1092	dbSNP
rs1031465124	1099	dbSNP
rs1270221823	1102	dbSNP
rs892584012	1103	dbSNP
rs530762984	1107	dbSNP
rs1279698176	1109	dbSNP
rs367614489	1114	dbSNP
rs1231587684	1115	dbSNP
rs1344576892	1118	dbSNP
rs1293902048	1119	dbSNP
rs1459343913	1121	dbSNP
rs1274450265	1122	dbSNP
rs1465140356	1124	dbSNP
rs1424505406	1126	dbSNP
rs1196099201	1128	dbSNP
rs1479586211	1134	dbSNP
rs867904909	1141	dbSNP
rs1192166194	1144	dbSNP
rs1489724325	1155	dbSNP
rs1231645262	1165	dbSNP
rs184653314	1166	dbSNP
rs934707405	1172	dbSNP
rs1272581269	1192	dbSNP
rs558629854	1198	dbSNP
rs1322436954	1229	dbSNP
rs1388274711	1245	dbSNP
rs1028589499	1250	dbSNP
rs1447495630	1258	dbSNP
rs756456098	1263	dbSNP
rs952957182	1266	dbSNP
rs778443160	1267	dbSNP
rs1177114577	1272	dbSNP
rs984737349	1286	dbSNP
rs1449614760	1287	dbSNP
rs1197927236	1288	dbSNP
rs917372303	1289	dbSNP
rs1458612397	1302	dbSNP
rs1239426881	1303	dbSNP
rs570932075	1309	dbSNP
rs1450945360	1310	dbSNP
rs1166397743	1313	dbSNP
rs189427997	1327	dbSNP
rs961447944	1329	dbSNP
rs992573171	1337	dbSNP
rs1389000855	1344	dbSNP
rs916975010	1349	dbSNP
rs948402121	1353	dbSNP
rs1164744280	1360	dbSNP
rs764378203	1365	dbSNP
rs34882908	1367	dbSNP
rs1308276256	1371	dbSNP
rs754243903	1373	dbSNP
rs905750894	1378	dbSNP
rs541773408	1390	dbSNP
rs757656799	1393	dbSNP
rs1049334050	1396	dbSNP
rs1467789776	1399	dbSNP
rs1158455860	1440	dbSNP
rs182204054	1441	dbSNP
rs1382678773	1443	dbSNP
rs1295736471	1445	dbSNP
rs1182335896	1454	dbSNP
rs1004714906	1471	dbSNP
rs372559137	1482	dbSNP
rs901630890	1486	dbSNP
rs891611010	1488	dbSNP
rs1284991414	1490	dbSNP
rs1217087271	1499	dbSNP
rs1355645043	1503	dbSNP
rs1312789190	1511	dbSNP
rs995781321	1512	dbSNP
rs779505990	1520	dbSNP
rs954412460	1538	dbSNP
rs1354181005	1539	dbSNP
rs1397969106	1545	dbSNP
rs1383105675	1549	dbSNP
rs1208948166	1550	dbSNP
rs374053277	1565	dbSNP
rs979045649	1569	dbSNP
rs1041167884	1576	dbSNP
rs541162646	1577	dbSNP
rs1189054660	1579	dbSNP
rs1426312255	1583	dbSNP
rs111858017	1594	dbSNP
rs901225533	1598	dbSNP
rs1186258198	1604	dbSNP
rs1247074579	1618	dbSNP
rs564632056	1620	dbSNP
rs1208384403	1622	dbSNP
rs1489144836	1625	dbSNP
rs996854601	1627	dbSNP
rs1370649775	1628	dbSNP
rs917319833	1638	dbSNP
rs746474413	1639	dbSNP
rs950130375	1642	dbSNP
rs1338572150	1643	dbSNP
rs1277802754	1644	dbSNP
rs1400904523	1646	dbSNP
rs1389827812	1651	dbSNP
rs980068432	1656	dbSNP
rs144563855	1657	dbSNP
rs1392109377	1659	dbSNP
rs1318734691	1662	dbSNP
rs546001875	1663	dbSNP
rs1453386690	1667	dbSNP
rs1170879341	1669	dbSNP
rs930514448	1676	dbSNP
rs1314946746	1683	dbSNP
rs1048971901	1691	dbSNP
rs1243959749	1702	dbSNP
rs886391145	1710	dbSNP
rs148004038	1714	dbSNP
rs1316133463	1718	dbSNP
rs1257517034	1720	dbSNP
rs1219408064	1722	dbSNP
rs1040208700	1725	dbSNP
rs7092702	1730	dbSNP
rs1332077754	1738	dbSNP
rs1326099782	1739	dbSNP
rs7076705	1745	dbSNP
rs1398840774	1749	dbSNP
rs992929620	1750	dbSNP
rs1331480647	1753	dbSNP
rs1029042508	1754	dbSNP
rs1422598353	1758	dbSNP
rs773080700	1759	dbSNP
rs1461505031	1762	dbSNP
rs1379212828	1769	dbSNP
rs368620011	1778	dbSNP
rs1179501755	1785	dbSNP
rs1457264946	1795	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.