>HMOX1|NM_002133|3'UTR
   1 ATGCAGGCATGCTGGCTCCCAGGGCCATGAACTTTGTCCGGTGGAAGGCCTTCTTTCTAGAGAGGGAATTCTCTTGGCTG
  81 GCTTCCTTACCGTGGGCACTGAAGGCTTTCAGGGCCTCCAGCCCTCTCACTGTGTCCCTCTCTCTGGAAAGGAGGAAGGA
 161 GCCTATGGCATCTTCCCCAACGAAAAGCACATCCAGGCAATGGCCTAAACTTCAGAGGGGGCGAAGGGATCAGCCCTGCC
 241 CTTCAGCATCCTCAGTTCCTGCAGCAGAGCCTGGAAGACACCCTAATGTGGCAGCTGTCTCAAACCTCCAAAAGCCCTGA
 321 GTTTCAAGTATCCTTGTTGACACGGCCATGACCACTTTCCCCGTGGGCCATGGCAATTTTTACACAAACCTGAAAAGATG
 401 TTGTGTCTTGTGTTTTTGTCTTATTTTTGTTGGAGCCACTCTGTTCCTGGCTCAGCCTCAAATGCAGTATTTTTGTTGTG
 481 TTCTGTTGTTTTTATAGCAGGGTTGGGGTGGTTTTTGAGCCATGCGTGGGTGGGGAGGGAGGTGTTTAACGGCACTGTGG
 561 CCTTGGTCTAACTTTTGTGTGAAATAATAAACAACATTGTCTGATAGTAGCTTGAAAAAAAAAAAAAAAAAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ucuuuCAUGAAGGGAGACCUc 5' || | ||:||||||| Target 5' actgtGTCCCTCTCTCTGGAa 3'	129 - 149	160.00	-14.60
2	miRNA 3' ucUUUCAUGAAGGGAGACCUc 5' :||| ||||::||| || Target 5' tgGAAGGCCTTCTTTCTAGAg 3'	42 - 62	123.00	-17.90
3	miRNA 3' ucUUUCAUGAA-----GGGAGACCuc 5' :|:||::|| | || ||| Target 5' ggGAGGTGTTTAACGGCACTGTGGcc 3'	537 - 562	103.00	-15.20

Mutant ID	Mutant Position	Mutant Source
COSN30472607	20	COSMIC
COSN31572909	30	COSMIC
COSN31491163	41	COSMIC
COSN31581937	48	COSMIC
COSN30114081	54	COSMIC
COSN30494176	55	COSMIC
COSN30105902	84	COSMIC
COSN30473218	92	COSMIC
COSN30100557	147	COSMIC
COSN31589901	204	COSMIC
COSN4785198	311	COSMIC
COSN19382191	374	COSMIC
COSN20783310	393	COSMIC
COSN31538655	452	COSMIC
COSN1261306	468	COSMIC
COSN25669819	468	COSMIC
COSN26551912	531	COSMIC
COSN31490521	551	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs774924065	3	dbSNP
rs1446761731	4	dbSNP
rs748669503	5	dbSNP
rs1314380699	8	dbSNP
rs1279625350	9	dbSNP
rs1190625712	12	dbSNP
rs367987395	13	dbSNP
rs773615991	15	dbSNP
rs1477674942	21	dbSNP
rs1381489947	23	dbSNP
rs530752316	26	dbSNP
rs1198846678	27	dbSNP
rs1239513778	28	dbSNP
rs1456057349	29	dbSNP
rs1178012514	30	dbSNP
rs995042284	36	dbSNP
rs866643988	39	dbSNP
rs201438906	40	dbSNP
rs776640266	41	dbSNP
rs894731419	49	dbSNP
rs760436375	51	dbSNP
rs759671776	52	dbSNP
rs550546401	54	dbSNP
rs1168602266	59	dbSNP
rs989347198	63	dbSNP
rs149739243	65	dbSNP
rs559481884	72	dbSNP
rs1446583871	75	dbSNP
rs539113031	76	dbSNP
rs753850950	82	dbSNP
rs1184856778	87	dbSNP
rs977151022	92	dbSNP
rs906139651	93	dbSNP
rs749455128	94	dbSNP
rs1206028066	96	dbSNP
rs1344383588	97	dbSNP
rs957403922	99	dbSNP
rs1048795422	102	dbSNP
rs1172733959	104	dbSNP
rs990017315	105	dbSNP
rs1314543934	107	dbSNP
rs1412867220	113	dbSNP
rs1354819056	126	dbSNP
rs1308550658	130	dbSNP
rs757798115	133	dbSNP
rs111787422	134	dbSNP
rs779325381	136	dbSNP
rs1337975166	139	dbSNP
rs1408921678	144	dbSNP
rs886147364	145	dbSNP
rs1167633638	146	dbSNP
rs147862586	150	dbSNP
rs1019162710	152	dbSNP
rs1183496131	154	dbSNP
rs566260020	156	dbSNP
rs1286578268	166	dbSNP
rs1484095562	169	dbSNP
rs911889128	173	dbSNP
rs901692220	178	dbSNP
rs944731148	182	dbSNP
rs1036389777	183	dbSNP
rs1297196577	185	dbSNP
rs1231290748	189	dbSNP
rs535207353	191	dbSNP
rs1333838488	202	dbSNP
rs1222661324	205	dbSNP
rs1418000413	212	dbSNP
rs1263756447	214	dbSNP
rs1305590722	215	dbSNP
rs967971070	217	dbSNP
rs1392505172	221	dbSNP
rs978729173	222	dbSNP
rs758713757	223	dbSNP
rs1394156483	224	dbSNP
rs371438796	234	dbSNP
rs930769568	239	dbSNP
rs17880056	245	dbSNP
rs894671137	254	dbSNP
rs1471409631	262	dbSNP
rs1362144986	271	dbSNP
rs1213798868	272	dbSNP
rs1181269260	275	dbSNP
rs11555832	279	dbSNP
rs1212012751	283	dbSNP
rs1467884197	288	dbSNP
rs1268459209	295	dbSNP
rs112611068	312	dbSNP
rs906855760	326	dbSNP
rs1348537073	327	dbSNP
rs1278870084	329	dbSNP
rs1160540251	332	dbSNP
rs1233400308	333	dbSNP
rs769322032	337	dbSNP
rs370298007	344	dbSNP
rs557349117	345	dbSNP
rs1321565482	346	dbSNP
rs1402806625	347	dbSNP
rs577322782	349	dbSNP
rs957145319	363	dbSNP
rs1158477491	364	dbSNP
rs572339648	366	dbSNP
rs1011790497	368	dbSNP
rs1158969918	369	dbSNP
rs1396911516	371	dbSNP
rs1191997199	375	dbSNP
rs930923547	376	dbSNP
rs181746701	381	dbSNP
rs772833576	390	dbSNP
rs954051818	396	dbSNP
rs907546802	402	dbSNP
rs986660564	402	dbSNP
rs1283523306	404	dbSNP
rs1200924451	411	dbSNP
rs559862155	411	dbSNP
rs1343253245	413	dbSNP
rs912505747	421	dbSNP
rs1404155818	436	dbSNP
rs748685954	444	dbSNP
rs186402981	447	dbSNP
rs1277499426	450	dbSNP
rs771027320	454	dbSNP
rs1233115841	459	dbSNP
rs1342521052	470	dbSNP
rs972044433	476	dbSNP
rs565313803	480	dbSNP
rs1040102921	481	dbSNP
rs1325019256	482	dbSNP
rs901617332	486	dbSNP
rs1325352638	495	dbSNP
rs1377354242	496	dbSNP
rs1222034512	501	dbSNP
rs1171920320	508	dbSNP
rs995957879	516	dbSNP
rs1169839115	520	dbSNP
rs530826190	521	dbSNP
rs930738871	524	dbSNP
rs1049653060	526	dbSNP
rs1486574242	527	dbSNP
rs1255317143	529	dbSNP
rs775963356	532	dbSNP
rs916063716	533	dbSNP
rs1342229214	534	dbSNP
rs948896029	536	dbSNP
rs1045987145	545	dbSNP
rs759528071	551	dbSNP
rs192434406	552	dbSNP
rs199990093	557	dbSNP
rs1177150566	560	dbSNP
rs775144949	562	dbSNP
rs971507747	569	dbSNP
rs5755721	578	dbSNP
rs530785590	584	dbSNP
rs1331158003	589	dbSNP
rs1400854196	594	dbSNP
rs1052795368	596	dbSNP
rs892826613	598	dbSNP
rs1163693542	601	dbSNP
rs113172420	605	dbSNP
rs1028512414	609	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.

- Genes & development, 2013

When adapting to environmental stress, cells attenuate and reprogram their translational output. In part, these altered translation profiles are established through changes in the interactions between RNA-binding proteins and mRNAs. The Argonaute 2 (Ago2)/microRNA (miRNA) machinery has been shown to participate in stress-induced translational up-regulation of a particular mRNA, CAT-1; however, a detailed, transcriptome-wide understanding of the involvement of Ago2 in the process has been lacking. Here, we profiled the overall changes in Ago2-mRNA interactions upon arsenite stress by cross-linking immunoprecipitation (CLIP) followed by high-throughput sequencing (CLIP-seq). Ago2 displayed a significant remodeling of its transcript occupancy, with the majority of 3' untranslated region (UTR) and coding sequence (CDS) sites exhibiting stronger interaction. Interestingly, target sites that were destined for release from Ago2 upon stress were depleted in miRNA complementarity signatures, suggesting an alternative mode of interaction. To compare the changes in Ago2-binding patterns across transcripts with changes in their translational states, we measured mRNA profiles on ribosome/polysome gradients by RNA sequencing (RNA-seq). Increased Ago2 occupancy correlated with stronger repression of translation for those mRNAs, as evidenced by a shift toward lighter gradient fractions upon stress, while release of Ago2 was associated with the limited number of transcripts that remained translated. Taken together, these data point to a role for Ago2 and the mammalian miRNAs in mediating the translational component of the stress response.