>SLC1A5|NM_001145144|3'UTR
   1 ACCCCGGGAGGGACCTTCCCTGCCCTGCTGGGGGTGCTCTTTGGACACTGGATTATGAGGAATGGATAAATGGATGAGCT
  81 AGGGCTCTGGGGGTCTGCCTGCACACTCTGGGGAGCCAGGGGCCCCAGCACCCTCCAGGACAGGAGATCTGGGATGCCTG
 161 GCTGCTGGAGTACATGTGTTCACAAGGGTTACTCCTCAAAACCCCCAGTTCTCACTCATGTCCCCAACTCAAGGCTAGAA
 241 AACAGCAAGATGGAGAAATAATGTTCTGCTGCGTCCCCACCGTGACCTGCCTGGCCTCCCCTGTCTCAGGGAGCAGGTCA
 321 CAGGTCACCATGGGGAATTCTAGCCCCCACTGGGGGGATGTTACAACACCATGCTGGTTATTTTGGCGGCTGTAGTTGTG
 401 GGGGGATGTGTGTGTGCACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCTGTGACCTCCTGTCCCCATGG
 481 TACGTCCCACCCTGTCCCCAGATCCCCTATTCCCTCCACAATAACAGAAACACTCCCAGGGACTCTGGGGAGAGGCTGAG
 561 GACAAATACCTGCTGTCACTCCAGAGGACATTTTTTTTAGCAATAAAATTGAGTGTCAACTATTTAA

ID	Duplex structure	Position	Score	MFE
1	miRNA 3' gaCGCCUUUCU-----C-UUCGUGCACc 5' |:||:::|| | : ||||||| Target 5' ttGTGGGGGGATGTGTGTGTGCACGTGt 3'	396 - 423	148.00	-21.80
2	miRNA 3' gaCGCCUUUCUC-UUCGUGCACc 5' || | :||| | ||:||| Target 5' tgGCTGCTGGAGTA--CATGTGt 3'	159 - 179	107.00	-14.50
3	miRNA 3' gacgccuuucucuUCGUGCAcc 5' :|:|||| Target 5' ctcctgtccccatGGTACGTcc 3'	466 - 487	105.00	-7.16

Mutant ID	Mutant Position	Mutant Source
COSN30139477	2	COSMIC
COSN30717183	5	COSMIC
COSN31600850	6	COSMIC
COSN30525265	19	COSMIC
COSN31493057	20	COSMIC
COSN30513356	29	COSMIC
COSN30130209	69	COSMIC
COSN30464003	91	COSMIC
COSN31510557	99	COSMIC
COSN30502969	106	COSMIC
COSN30181003	135	COSMIC
COSN22476874	211	COSMIC
COSN6145609	356	COSMIC
COSN23527959	388	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1294270631	2	dbSNP
rs1382253740	3	dbSNP
rs774538305	4	dbSNP
rs529969003	5	dbSNP
rs749445334	6	dbSNP
rs1405868357	8	dbSNP
rs1177559354	9	dbSNP
rs1471094395	10	dbSNP
rs773479751	11	dbSNP
rs1372044580	12	dbSNP
rs769987963	14	dbSNP
rs1429127279	15	dbSNP
rs748169342	18	dbSNP
rs1397426234	22	dbSNP
rs1367372212	23	dbSNP
rs1012474872	25	dbSNP
rs895261202	28	dbSNP
rs1460801031	30	dbSNP
rs1048948513	31	dbSNP
rs780156575	32	dbSNP
rs1337066298	33	dbSNP
rs377465288	34	dbSNP
rs771858328	40	dbSNP
rs1384165667	45	dbSNP
rs745711019	46	dbSNP
rs1319192502	47	dbSNP
rs778544872	48	dbSNP
rs560908326	54	dbSNP
rs897170954	57	dbSNP
rs62134839	59	dbSNP
rs944312611	65	dbSNP
rs1276610846	69	dbSNP
rs1348506399	72	dbSNP
rs912790304	79	dbSNP
rs1233417286	94	dbSNP
rs1414005292	96	dbSNP
rs1489132785	101	dbSNP
rs1205409320	106	dbSNP
rs1353329925	120	dbSNP
rs1260596553	123	dbSNP
rs1314703519	129	dbSNP
rs1444848827	135	dbSNP
rs1448645914	147	dbSNP
rs371673136	160	dbSNP
rs1253018308	164	dbSNP
rs901214366	170	dbSNP
rs932806897	177	dbSNP
rs1408434968	181	dbSNP
rs1041437498	186	dbSNP
rs921744848	190	dbSNP
rs1475108240	207	dbSNP
rs564639315	215	dbSNP
rs1380674556	219	dbSNP
rs989229957	221	dbSNP
rs1371773890	225	dbSNP
rs1440136986	227	dbSNP
rs957739063	228	dbSNP
rs911284368	236	dbSNP
rs1318566297	237	dbSNP
rs1483840485	239	dbSNP
rs769333918	251	dbSNP
rs930490496	253	dbSNP
rs1201749021	254	dbSNP
rs1211215970	258	dbSNP
rs1273301944	262	dbSNP
rs143229941	263	dbSNP
rs576215661	273	dbSNP
rs1232273870	274	dbSNP
rs1332481316	281	dbSNP
rs73566928	282	dbSNP
rs1034698891	296	dbSNP
rs1451941832	300	dbSNP
rs981704800	302	dbSNP
rs1395380329	303	dbSNP
rs969130290	303	dbSNP
rs1297695569	311	dbSNP
rs35076856	316	dbSNP
rs1023312093	317	dbSNP
rs1010599069	323	dbSNP
rs1332355890	333	dbSNP
rs1012115456	337	dbSNP
rs748117764	340	dbSNP
rs542885033	346	dbSNP
rs1391841053	350	dbSNP
rs113044612	352	dbSNP
rs1333726322	358	dbSNP
rs1392775990	362	dbSNP
rs1234791793	366	dbSNP
rs534714866	370	dbSNP
rs1273400141	371	dbSNP
rs1026740741	377	dbSNP
rs995564030	385	dbSNP
rs139875462	387	dbSNP
rs760442190	388	dbSNP
rs897223044	389	dbSNP
rs1037019762	393	dbSNP
rs944030393	401	dbSNP
rs554098570	402	dbSNP
rs1481912608	405	dbSNP
rs936559265	406	dbSNP
rs181186987	408	dbSNP
rs891413292	410	dbSNP
rs1050430918	416	dbSNP
rs1031587228	417	dbSNP
rs932878552	420	dbSNP
rs1418846117	421	dbSNP
rs1474966191	425	dbSNP
rs1238233418	426	dbSNP
rs778948051	427	dbSNP
rs780342287	427	dbSNP
rs749164545	429	dbSNP
rs750711937	429	dbSNP
rs1212711014	430	dbSNP
rs1462460759	431	dbSNP
rs901389862	431	dbSNP
rs1041083415	433	dbSNP
rs1006865850	434	dbSNP
rs1416375968	435	dbSNP
rs889776229	435	dbSNP
rs1266997543	437	dbSNP
rs1231010107	450	dbSNP
rs1221958998	454	dbSNP
rs1297122959	456	dbSNP
rs1186028740	458	dbSNP
rs1228858837	458	dbSNP
rs1251235076	458	dbSNP
rs1323283367	458	dbSNP
rs1394399577	458	dbSNP
rs1429175956	458	dbSNP
rs1436433858	458	dbSNP
rs143976687	458	dbSNP
rs147659556	458	dbSNP
rs1482318749	458	dbSNP
rs55753437	458	dbSNP
rs59359060	458	dbSNP
rs71340612	458	dbSNP
rs758231406	458	dbSNP
rs765224486	458	dbSNP
rs79749608	458	dbSNP
rs878892310	458	dbSNP
rs1290248288	459	dbSNP
rs1313272677	459	dbSNP
rs1362118315	459	dbSNP
rs751841442	459	dbSNP
rs776529948	459	dbSNP
rs1213475072	471	dbSNP
rs1048532658	477	dbSNP
rs1340679263	479	dbSNP
rs931382330	480	dbSNP
rs190264892	483	dbSNP
rs143857841	484	dbSNP
rs1208344862	489	dbSNP
rs940621534	505	dbSNP
rs558276473	507	dbSNP
rs1244044624	509	dbSNP
rs1379238014	514	dbSNP
rs1486272583	516	dbSNP
rs1186004300	521	dbSNP
rs1246202911	526	dbSNP
rs1455547767	527	dbSNP
rs927847826	529	dbSNP
rs1394309208	530	dbSNP
rs1310022722	535	dbSNP
rs1432620372	540	dbSNP
rs936422020	547	dbSNP
rs1160925466	550	dbSNP
rs377412405	551	dbSNP
rs1345427361	561	dbSNP
rs117158128	569	dbSNP
rs1429524786	569	dbSNP
rs969056658	575	dbSNP
rs1443806815	576	dbSNP
rs1304776435	577	dbSNP
rs1370797045	588	dbSNP
rs1217827274	589	dbSNP
rs1317754960	590	dbSNP
rs1215267449	594	dbSNP
rs1266345434	612	dbSNP

- Nature, 2009

MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein-RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein-RNA complexes in mouse brain. This produced two simultaneous data sets-Ago-miRNA and Ago-mRNA binding sites-that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA-mRNA interactions.

- 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.

- Breast cancer research and treatment, 2014

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.