ID	Duplex structure	Position	Score	MFE
1	miRNA 3' cgaggucaGAGCUGCA---AGGGCccc 5' :|| |:|| |||:| Target 5' agaaagcaTTCAATGTCAGTCCTGttt 3'	129 - 155	96.00	-8.05
2	miRNA 3' cgAGGUCAGAGCUGCAAGGGCccc 5' |:|| | | | ||::| Target 5' agTTCAAAAT-GCAGATCTTGcca 3'	102 - 124	73.00	-7.12
3	miRNA 3' cgAGGUCAG-AG-CUG-CAAGGGcccc 5' |:|| || || ||| | ||| Target 5' caTTCA-TCATCAGACAG--CCCacac 3'	24 - 47	68.00	-9.16

Mutant ID	Mutant Position	Mutant Source
COSN28665542	1	COSMIC
COSN8605702	1	COSMIC
COSN27006262	4	COSMIC
COSN13802004	5	COSMIC
COSN20062536	8	COSMIC
COSN8866972	15	COSMIC
COSN30526208	16	COSMIC
COSN27006260	19	COSMIC
COSN27006264	22	COSMIC
COSN30158165	23	COSMIC
COSN30488072	28	COSMIC
COSN30483809	31	COSMIC
COSN30122169	52	COSMIC
COSN13802002	58	COSMIC
COSN27006267	59	COSMIC
COSN31595456	64	COSMIC
COSN30506461	79	COSMIC
COSN32074036	80	COSMIC
COSN30535826	86	COSMIC
COSN20066950	90	COSMIC
COSN31546517	99	COSMIC
COSN30176793	106	COSMIC
COSN30451413	107	COSMIC
COSN8605701	145	COSMIC
COSN30193459	150	COSMIC
COSN31613500	174	COSMIC
COSN17076718	189	COSMIC
COSN1215145	190	COSMIC
COSN19614732	190	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs1293259681	2	dbSNP
rs752193334	3	dbSNP
rs764657811	4	dbSNP
rs377322858	5	dbSNP
rs776455500	11	dbSNP
rs1301543277	13	dbSNP
rs1291	15	dbSNP
rs566498803	16	dbSNP
rs773059860	18	dbSNP
rs547878261	19	dbSNP
rs189601956	20	dbSNP
rs57172286	21	dbSNP
rs768653510	22	dbSNP
rs1366265810	25	dbSNP
rs1297133298	26	dbSNP
rs968491912	30	dbSNP
rs749430591	33	dbSNP
rs1373262084	40	dbSNP
rs1294069721	41	dbSNP
rs1457311520	46	dbSNP
rs117141874	47	dbSNP
rs991462583	65	dbSNP
rs931559062	66	dbSNP
rs749614011	76	dbSNP
rs991201458	77	dbSNP
rs199908753	78	dbSNP
rs1248364970	79	dbSNP
rs1197432396	85	dbSNP
rs1253934557	91	dbSNP
rs867511706	104	dbSNP
rs1339538190	119	dbSNP
rs1254868835	122	dbSNP
rs1401743189	130	dbSNP
rs959509545	141	dbSNP
rs1035165762	152	dbSNP
rs1003779602	162	dbSNP
rs184630429	163	dbSNP
rs1303611443	173	dbSNP
rs1235483977	176	dbSNP
rs1335497724	181	dbSNP
rs1019647173	182	dbSNP
rs986846948	185	dbSNP
rs1397387946	187	dbSNP
rs1310751677	188	dbSNP
rs200218055	190	dbSNP
rs1227609115	191	dbSNP
rs954163207	191	dbSNP

- Nature methods, 2011

Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.

- 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.

- Genome research, 2016

DNA damage activates TP53-regulated surveillance mechanisms that are crucial in suppressing tumorigenesis. TP53 orchestrates these responses directly by transcriptionally modulating genes, including microRNAs (miRNAs), and by regulating miRNA biogenesis through interacting with the DROSHA complex. However, whether the association between miRNAs and AGO2 is regulated following DNA damage is not yet known. Here, we show that, following DNA damage, TP53 interacts with AGO2 to induce or reduce AGO2's association of a subset of miRNAs, including multiple let-7 family members. Furthermore, we show that specific mutations in TP53 decrease rather than increase the association of let-7 family miRNAs, reducing their activity without preventing TP53 from interacting with AGO2. This is consistent with the oncogenic properties of these mutants. Using AGO2 RIP-seq and PAR-CLIP-seq, we show that the DNA damage-induced increase in binding of let-7 family members to the RISC complex is functional. We unambiguously determine the global miRNA-mRNA interaction networks involved in the DNA damage response, validating them through the identification of miRNA-target chimeras formed by endogenous ligation reactions. We find that the target complementary region of the let-7 seed tends to have highly fixed positions and more variable ones. Additionally, we observe that miRNAs, whose cellular abundance or differential association with AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits. TP53-mediated regulation of AGO2-miRNA interaction represents a new mechanism of miRNA regulation in carcinogenesis.

- Neoplasia (New York, N.Y.), 2016

MicroRNA (miRNA) deregulation in prostate cancer (PCa) contributes to PCa initiation and metastatic progression. To comprehensively define the cancer-associated changes in miRNA targeting and function in commonly studied models of PCa, we performed photoactivatable ribonucleoside-enhanced cross-linking immunoprecipitation of the Argonaute protein in a panel of PCa cell lines modeling different stages of PCa progression. Using this comprehensive catalogue of miRNA targets, we analyzed miRNA targeting on known drivers of PCa and examined tissue-specific and stage-specific pathway targeting by miRNAs. We found that androgen receptor is the most frequently targeted PCa oncogene and that miR-148a targets the largest number of known PCa drivers. Globally, tissue-specific and stage-specific changes in miRNA targeting are driven by homeostatic response to active oncogenic pathways. Our findings indicate that, even in advanced PCa, the miRNA pool adapts to regulate continuing alterations in the cancer genome to balance oncogenic molecular changes. These findings are important because they are the first to globally characterize miRNA changes in PCa and demonstrate how the miRNA target spectrum responds to staged tumorigenesis.