Shivendra Kishore

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins

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.

The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing

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.

Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression

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.

Insights into snoRNA biogenesis and processing from PAR-CLIP of snoRNA core proteins and small RNA sequencing

BackgroundIn 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.ResultsAnalysis 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.ConclusionsWe 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.

Rapid generation of splicing reporters with pSpliceExpress.

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.

Modeling the binding specificity of the RNA-binding protein GLD-1 suggests a function of coding region–located sites in translational repression

To understand the function of the hundreds of RNA-binding proteins (RBPs) that are encoded in animal genomes it is important to identify their target RNAs. Although it is generally accepted that the binding specificity of an RBP is well described in terms of the nucleotide sequence of its binding sites, other factors such as the structural accessibility of binding sites or their clustering, to enable binding of RBP multimers, are also believed to play a role. Here we focus on GLD-1, a translational regulator of Caenorhabditis elegans, whose binding specificity and targets have been studied with a variety of methods such as CLIP (cross-linking and immunoprecipitation), RIP-Chip (microarray measurement of RNAs associated with an immunoprecipitated protein), profiling of polysome-associated mRNAs and biophysical determination of binding affinities of GLD-1 for short nucleotide sequences. We show that a simple biophysical model explains the binding of GLD-1 to mRNA targets to a large extent, and that taking into account the accessibility of putative target sites significantly improves the prediction of GLD-1 binding, particularly due to a more accurate prediction of binding in transcript coding regions. Relating GLD-1 binding to translational repression and stabilization of its target transcripts we find that binding sites along the entire transcripts contribute to functional responses, and that CDS-located sites contribute most to translational repression. Finally, biophysical measurements of GLD-1 affinity for a small number of oligonucleotides appear to allow an accurate reconstruction of the sequence specificity of the protein. This approach can be applied to uncover the specificity and function of other RBPs.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages

Background 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). Methods 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.

Role of Alternative Splicing of the 5-HT2C Receptor in the Prader–Willi Syndrome

The 5-HT2C receptor (5-HT2CR) belongs to the family of seven transmembrane-containing G-protein-coupled receptors (GPCRs) and is located on the human X chromosome band q24. The pre-mRNA generated from this gene undergoes extensive RNA processing. Due to RNA editing and alternative splicing, the gene could generate at least 288 different isoforms. Under physiological conditions, the RNA processing is tightly regulated. Deviations from the normal processing patterns that disturb the balance between the mRNA isoforms lead to diseases. Here we describe the regulation of alternative splicing of exon Vb of the 5-HT2C receptor. This exon encodes the second intracellular loop that is important for signaling. The exon is regulated by a splicing silencer that can be modified by RNA editing. The inclusion of the nonedited exon Vb is promoted by a small nucleolar RNA (snoRNA), HBII-52/SNORD115 and its processed isoforms. The regulation of this exon appears to be altered in the Prader–Willi syndrome (PWS).

Ewing sarcoma breakpoint region 1 prevents transcription-associated genome instability

Ewing Sarcoma break point region 1 (EWSR1) is a multi-functional RNA-binding protein that is involved in many cellular processes, from gene expression to RNA processing and transport. Translocations into its locus lead to chimeric proteins with tumorigenic activity, that lack the RNA binding domain. With crosslinking and immunoprecipitation we have found that EWSR1 binds to intronic regions that are present in polyadenylated nuclear RNAs, which include the translocation-prone region of its own locus. Reduced EWSR1 expression leads to gene expression changes that indicate reduced proliferation. By fluorescence in situ hybridization (FISH) with break-apart probes that flanked the translocation-prone region within the EWSR1 locus we found that reduced EWSR1 expression increases the frequency of split signals, indicative of DNA double strand breaks (DSB). The response in phosphorylated histone H2AX and p53-binding protein 1 (53BP1) double-stained foci to the topoisomerase poison camptothecin in cells treated with a control shRNA and with sh-EWSR1 further suggests that EWSR1 functions in the prevention of DNA DSBs. Our data reveal a new function of the EWSR1 member of the FET family and suggest a connection between the RNA-binding activity of EWSR1 and the instability of its own locus that may play a role in malignancy-associated translocations.

Analysis of Alternative Splicing with Microarrays

Alternative splicing is one of the most important post-transcriptional processing steps that enhances genomic information by generating multiple RNA isoforms from a single gene. Recently, microarrays have been developed that can detect changes in splice site selection. Currently, the biggest challenge for the analysis of alternative splicing with microarrays is the bioinformatics analysis of array data and their low reproducibility by RT-PCR. Despite these problems, microarrays revealed an unexpected number of expressed RNAs, showed changes of alternative splicing in diseases and indicated that a splicing factor regulates a biologically meaningful set of genes.