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1.  HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages 
PLoS ONE  2015;10(7):e0132127.
MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM).
The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.
Results and Conclusion
PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.
PMCID: PMC4520458  PMID: 26226348
2.  Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing 
Genome Biology  2013;14(5):R45.
In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequence long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins.
Analysis of these data sets reveals that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently cataloged. We further find that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs reveals that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein.
We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon.
PMCID: PMC4053766  PMID: 23706177
3.  Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression 
Briefings in Functional Genomics  2010;9(5-6):391-404.
Eukaryotic cells express a large variety of ribonucleic acid-(RNA)-binding proteins (RBPs) with diverse affinity and specificity towards target RNAs that play a crucial role in almost every aspect of RNA metabolism. In addition, specific domains in RBPs impart catalytic activity or mediate protein–protein interactions, making RBPs versatile regulators of gene expression. In this review, we elaborate on recent experimental and computational approaches that have increased our understanding of RNA–protein interactions and their role in cellular function. We review aspects of gene expression that are modulated post-transcriptionally by RBPs, namely the stability of polymerase II-derived mRNA transcripts and their rate of translation into proteins. We further highlight the extensive regulatory networks of RBPs that implement a combinatorial control of gene expression. Taking cues from the recent development in the field, we argue that understanding spatio-temporal RNA–protein association on a transcriptome level will provide invaluable and unexpected insights into the regulatory codes that define growth, differentiation and disease.
PMCID: PMC3080770  PMID: 21127008
RNA-binding proteins; RNA-binding domains; RBP–RNA interaction; RBP regulatory networks; RBP target identification
4.  The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing 
Human Molecular Genetics  2010;19(7):1153-1164.
The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader–Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.
PMCID: PMC2838533  PMID: 20053671
5.  Rapid generation of splicing reporters with pSpliceExpress 
Gene  2008;427(1-2):104.
Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo.
PMCID: PMC2821805  PMID: 18930792
Alternative splicing; High throughput; Recombination; Reporter gene

Results 1-5 (5)