Evolution, Function and Structure of the Splicing Factor PRPF39

Beside 5’-capping and 3’-polyadenylation, splicing is one of the essential steps in the processing of most protein-coding genes in higher eukaryotes. It is catalyzed by the spliceosome, a large and dynamic RNA-protein molecular machine that encompasses five core components, the U1, U2, U4, U5 and U6 snRNPs. For each splicing reaction, a spliceosome is assembled anew in a stepwise manner. Spliceosomes must accurately recognize each splice site, as a single mistake can result in the production of a non-functional and potentially toxic protein. The crucial step of exon definition is facilitated by the U1 and U2 snRNP during early splicing. Cryo electron microscopy structures of early spliceosomal complexes in yeast have shown that the Prp39/Prp42 heterodimer is a crucial scaffolding subcomplex. It acts as a hub for multiple protein-protein interactions for example the contact between the U1 and the U2 snRNP, indicating that the Prp39/Prp42 heterodimer is important for the precise spatial positioning of the U1 and U2 snRNPs relative to each other. Interestingly there is no homolog for Prp42 in higher eukaryotes. PRPF39 is largely unstudied in higher eukaryotes and came to our attention because it is alternatively spliced in a differential manner in murine naïve vs. memory T-cells. I could show that an alternative exon is included in a differential manner. This can control PRPF39 expression by NMD in a tissue- and activation-dependent manner in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. Furthermore, I solved the crystal structure of murine PRPF39 at 3.3 Å resolution. The protein is largely α-helical and the structure shows the protein to be organized as a homodimer. Dimerization in solution could be confirmed with further biophysical assays. The mode of PRPF39 homodimerization is strikingly similar to heterodimerization of Prp39 and Prp42 in yeast. Structure guided point mutations could completely abolish dimerization and by using the monomeric PRPF39 mutants I could show that the monomer has a detrimental effect on splicing in vitro. Based on a structural comparison of murine PRPF39 and the yeast heterodimer, we performed a phylogenetic analysis showing, that organisms with a Prp39 homodimer have a substantially shortened U1 snRNA compared to organisms with a Prp39/Prp42 heterodimer. Our analysis indicates that a shortened U1 snRNA accompanied by a PRPF39 homodimer was crucial in the evolutionary development of more complex splicing. This observation is unexpected, as fewer splicing factors usually mirror lower splicing complexity and not the opposite. Taken together, my results reveal the structural and functional implications of murine PRPF39 on splicing. The data suggests, that a PRPF39 homodimer acts to substitute the Prp39/Prp42 heterodimer observed in yeast. Additionally, the reduction in RNA and protein complexity of the U1 snRNP may have been crucial in allowing highly complex and sophisticated splicing regulation across species

evolution, structure and function of metazoan splicing factor PRPF39

In the yeast U1 snRNP the Prp39/Prp42 heterodimer is essential for early steps of spliceosome assembly. In metazoans no Prp42 ortholog exists, raising the question how the heterodimer is functionally substituted. Here we present the crystal structure of murine PRPF39, which forms a homodimer. Structure-guided point mutations disrupt dimer formation and inhibit splicing, manifesting the homodimer as functional unit. PRPF39 expression is controlled by NMD-inducing alternative splicing in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. A phylogenetic analysis reveals coevolution of shortened U1 snRNA and the absence of Prp42, which correlates with overall splicing complexity in different fungi. While current models correlate the diversity of spliceosomal proteins with splicing complexity, our study highlights a contrary case. We find that organisms with higher splicing complexity have substituted the Prp39/Prp42 heterodimer with a PRPF39 homodimer

AP-2α regulates migration of GN-11 neurons via a specific genetic programme involving the Axl receptor tyrosine kinase

<p>Abstract</p> <p>Background</p> <p>Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2α is a transcription factor essential for neural crest cell migration and its mutation results in apoptosis within this cell population, as demonstrated by genetic models.</p> <p>Results</p> <p>We down-modulated AP-2α expression in GN-11 neurons by RNA interference and observe reduced neuron migration following the activation of a specific genetic programme including the Adhesion Related Kinase (<it>Axl</it>) gene. We prove that <it>Axl </it>is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2α via the binding to one or more functional AP-2α binding sites present in its regulatory region. Analysis of migration in AP-2α null mouse embryo fibroblasts also reveals an essential role for AP-2α in cell movement via the activation of a distinct genetic programme.</p> <p>Conclusion</p> <p>We show that AP-2α plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2α regulated gene <it>Axl </it>is an essential player in GN-11 neuron migration.</p

Docker4Circ: A Framework for the Reproducible Characterization of circRNAs from RNA-Seq Data.

Recent improvements in cost-effectiveness of high-throughput technologies has allowed RNA sequencing of total transcriptomes suitable for evaluating the expression and regulation of circRNAs, a relatively novel class of transcript isoforms with suggested roles in transcriptional and post-transcriptional gene expression regulation, as well as their possible use as biomarkers, due to their deregulation in various human diseases. A limited number of integrated workflows exists for prediction, characterization, and differential expression analysis of circRNAs, none of them complying with computational reproducibility requirements. We developed Docker4Circ for the complete analysis of circRNAs from RNA-Seq data. Docker4Circ runs a comprehensive analysis of circRNAs in human and model organisms, including: circRNAs prediction; classification and annotation using six public databases; back-splice sequence reconstruction; internal alternative splicing of circularizing exons; alignment-free circRNAs quantification from RNA-Seq reads; and differential expression analysis. Docker4Circ makes circRNAs analysis easier and more accessible thanks to: (i) its R interface; (ii) encapsulation of computational tasks into docker images; (iii) user-friendly Java GUI Interface availability; and (iv) no need of advanced bash scripting skills for correct use. Furthermore, Docker4Circ ensures a reproducible analysis since all its tasks are embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project

Docker4Circ: A Framework for the Reproducible Characterization of circRNAs from RNA-Seq Data

Recent improvements in cost-effectiveness of high-throughput technologies has allowed RNA sequencing of total transcriptomes suitable for evaluating the expression and regulation of circRNAs, a relatively novel class of transcript isoforms with suggested roles in transcriptional and post-transcriptional gene expression regulation, as well as their possible use as biomarkers, due to their deregulation in various human diseases. A limited number of integrated workflows exists for prediction, characterization, and differential expression analysis of circRNAs, none of them complying with computational reproducibility requirements. We developed Docker4Circ for the complete analysis of circRNAs from RNA-Seq data. Docker4Circ runs a comprehensive analysis of circRNAs in human and model organisms, including: circRNAs prediction; classification and annotation using six public databases; back-splice sequence reconstruction; internal alternative splicing of circularizing exons; alignment-free circRNAs quantification from RNA-Seq reads; and differential expression analysis. Docker4Circ makes circRNAs analysis easier and more accessible thanks to: (i) its R interface; (ii) encapsulation of computational tasks into docker images; (iii) user-friendly Java GUI Interface availability; and (iv) no need of advanced bash scripting skills for correct use. Furthermore, Docker4Circ ensures a reproducible analysis since all its tasks are embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells

Effects of Oestrogen on MicroRNA Expression in Hormone-Responsive Breast Cancer Cells

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study