ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggAUGCAAGGUAUCAGAUGGu 5' ||| | : | | |||||| Target 5' acTAC-TGTGAAATTCTACCa 3'	76 - 95	122.00	-13.30
2	miRNA 3' ggaugcaAGGUAUCAGAUggu 5' |:|||| |||| Target 5' gaatctcTTCATAATCTAggc 3'	34 - 54	94.00	-5.30
3	miRNA 3' ggaUGCAAGGUAU--CAGAUGGU- 5' :| || :|| || ||:|: Target 5' ccaGCATTAAGTACTGTATATCGC 3'	93 - 116	92.00	-6.40

Mutant ID	Mutant Position	Mutant Source
COSN30115284	23	COSMIC
COSN30508507	40	COSMIC
COSN20084762	54	COSMIC
COSN5953468	100	COSMIC
COSN30164309	111	COSMIC
COSN30102240	114	COSMIC
COSN30140505	115	COSMIC
COSN31594324	158	COSMIC
COSN31598849	165	COSMIC
COSN29570420	217	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs368010022	3	dbSNP
rs1349071978	10	dbSNP
rs1222863148	27	dbSNP
rs374115487	30	dbSNP
rs763163739	31	dbSNP
rs1158192174	37	dbSNP
rs779872583	37	dbSNP
rs758633496	40	dbSNP
rs370355440	43	dbSNP
rs376276002	46	dbSNP
rs753252360	47	dbSNP
rs772194805	48	dbSNP
rs1161270550	49	dbSNP
rs1246802752	50	dbSNP
rs957087734	54	dbSNP
rs925697608	61	dbSNP
rs1236789727	64	dbSNP
rs1200881528	78	dbSNP
rs1466455934	81	dbSNP
rs548051805	96	dbSNP
rs1318993500	98	dbSNP
rs1394516877	107	dbSNP
rs977052895	110	dbSNP
rs73391838	112	dbSNP
rs1227050551	114	dbSNP
rs190992421	114	dbSNP
rs1358570106	115	dbSNP
rs1320004750	120	dbSNP
rs1024013024	125	dbSNP
rs997664673	129	dbSNP
rs1336983761	132	dbSNP
rs958679371	133	dbSNP
rs1419003201	139	dbSNP
rs1196724772	143	dbSNP
rs902092094	144	dbSNP
rs994631839	145	dbSNP
rs1192861988	151	dbSNP
rs1371733041	153	dbSNP
rs1041988048	157	dbSNP
rs1333567832	159	dbSNP
rs1164028482	161	dbSNP
rs946068541	165	dbSNP
rs1422821191	173	dbSNP
rs1448746193	174	dbSNP
rs914521567	192	dbSNP
rs1054467298	196	dbSNP
rs937391991	199	dbSNP
rs374481893	201	dbSNP
rs775566606	207	dbSNP
rs927377865	212	dbSNP
rs1403529022	218	dbSNP
rs1382728799	231	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.