December 11, 2017, Monday

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ASWorkshop Invited Lectures

Contents

Complete alternative splicing events

Michael Sammeth, Genome Bioinformatics Lab, Center for Genomics Regulation, Spain

Eukaryotic splicing structures are known to involve a high degree of alternative forms derived from a premature transcript by alternative splicing (AS). With the advent of new sequencing technologies, evidence for new splice forms becomes more and more easily available - bit by bit revealing that the true splicing diversity of AS events often comprises more than two alternatives and therefore cannot be sufficiently described by pairwise comparisons as conducted in analyzes hitherto. Further challenges emerge from the richness of data (millions of transcripts) and artifacts introduced during the technical process of obtaining transcript sequences (noise), especially when dealing with single-read sequences known as expressed sequence tags (ESTs).

I describe a method based on so-called splicing graphs to efficiently predict AS events in different resolutions (i.e., dimensions) from transcript annotations that allows for combination of fragmented EST data with full-length cDNAs and can cope with large datasets containing noise.

Applying this method to estimate the real complexity of alternative splicing, one can find thousands of novel AS events that either have been disregarded or mischaracterized in earlier works - e.g., events with the simultaneous skipping of 2 subsequent exons, with 2 alternative subsequent exons, with an alternative exon flanked by an exon flank variation upstream/downstream, etc....

Deeper bioinformatical analyzes on these events demonstrate that the exhaustive decomposition of alternative splicing variety into events indeed allows for drawing conclusions on the presumptive molecular mechanism that regulates events sharing a certain structure - and there are examples of wet lab experiments on specific genes that back up these hypotheses.

Furthermore, I show the potential of these events for instance to investigate AS in different gene sets, in the evolutionary context across several metazoan species and to retro-inspect the availability of evidence having become available over the recent years.


Evolution of splicing factors and signals in eukaryotes

Eduardo Eyras, Universitat Pompeu Fabra, Spain

SR proteins are essential for splicing in metazoans but are absent in yeast. By contrast, many fungi have SR protein homologs with variable arginine-rich regions analogous to the RS domain in metazoans.

We show that the density of RS repeats in these regions correlate with the conservation of the branch site signal, providing evidence for an ancestral origin of SR proteins and suggesting that the SR proteins and the branch site co-evolved. We propose that the expansion of RS repeats in SR proteins played a significant role in the relaxation of the splicing signals and possibly in the origin of regulated splicing.


Alternative splicing could explain the peculiar involvement of specific muscle groups in FSHD

Davide Gabellini, Università Vita-Salute San Raffaele

Facioscapulohumeral muscular dystrophy (FSHD) is the third most important hereditary disease affecting the skeletal muscle. Unlike the majority of genetic diseases, FSHD is not caused by mutation in a protein-coding gene but is the result of a complex epigenetic cascade activated by deletion of a 3.3 kb subtelomeric non-protein coding repeat (D4Z4) located on chromosome 4q35. Deletion of D4Z4 is associated to over-repression of several 4q35 genes. We have found that transgenic mice over-expressiong the 4q35 gene FRG1 display a disease with a variety of features characteristic of FSHD. Notably, in muscle of FRG1 transgenic mice and FSHD patients specific pre-mRNAs undergo aberrant alternative splicing.

The precise mechanism of action of FRG1 is unknown. FRG1 might bind RNA directly, and change splicing dynamic, or it might regulate the activity of splicing factors Understanding the role FRG1 plays in normal and in diseased muscle requires methods to identify the set of RNAs that FRG1 regulates in vivo and the use of an animal model of FSHD for RNA target validation. To address this aim systematically, we used splicing- sensitive microarrays to analyze RNA extracted from WT and FRG1 mice. We selected 40 genes for validation. RT-PCR analysis resulted in a very high validation of the microarray results. Interestingly, several observations indicate that the aberrant splicing in FRG1 transgenic mice was not a secondary effect of the dystrophic process. First, aberrant splicing could be detected in 4-week old FRG1 transgenic mice, a time at which histological and ultrastructural analysis revealed no evidence for muscular dystrophy. Second, the analyzed genes were spliced normally in mdx mice (the mouse model of Duchenne muscular dystrophy). Finally, altered splicing was also observed in C2C12 muscle cell cultures over-expressing FRG1, indicating that aberrant splicing was a direct and specific effect of FRG1 over-expression.

In FRG1 mice and FSHD patients, the disease affects a highly selective set of muscle groups. Hence, comparison of the splicing profile of muscle types differently affected should identify genes that might be involved in the different response to FRG1 over- expression. To this purpose, we compared the splicing profile of vastus lateralis and biceps brachii. Vastus lateralis is severely affected in FSHD patients and FRG1 mice, while biceps is mildly affected. Interestingly, we found that the degree of aberrant splicing accurately reflects the severity of disease in these two muscles.

Our results suggest that FSHD results from inappropriate over-expression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.


Pre mRNA splicing and disease: diagnostic aspects and basic mechanisms

Franco Pagani, International Centre for Genetic Engineering and Biotechnology

Pre mRNA splicing is a key step in the regulation of gene expression and due to its intrinsic complexity susceptible of pathological derangements. There are significant gaps in our knowledge of the precise molecular mechanisms of both constitutive and alternative splicing and characterization of splicing defects involved in human disease can provide novel information. Alterations leading to aberrant splicing events and the correct assessment of potentially disease-causing sequence changes are essential requirements for the design of specific therapeutic and diagnostic approaches.

We have characterised aberrant splicing events in several gene systems (cystic fibrosis, long QT syndrome, ataxia-telengectasia, BRCA1, FVII deficiency and Friedreich ataxia) that result from disruption of as yet unknown or poorly characterized splicing modulatory elements. The study of mutations in CFTR and BRCA1 exon identified Composite Exonic Regulatory Elements (CERES) and silencers binding to hnRNPA1/A2 and DAZAP1. Analysis in FVII deficiency provides novel therapeutic strategy to correct donor splice site defects using modified U1 snRNA and the study of GAA repeat expansions in Friedreich Ataxia highlight the importance of intronic regulatory elements in efficient processing of long introns. On the other hand, basic studied on processing of large intronic sequences using artificial cotranscriptional cleavage sequences (ribozymes) indicates a requirement for the continuity of the RNA molecule between intronic regulatory elements and alternatively spliced exons suggesting a possible role of miRNA in efficient processing of nascent transcript.


When a protein is a protein

Emerging functional and structural features from protein sequence analysis to validate putative proteins predicted by alternative splicing

Rita Casadio, Università degli Studi di Bologna

Alternative mRNA splicing enables genes to generate more than one gene product. Recently a detailed bioinformatics study of the alternatively spliced gene products annotated in the ENCODE pilot project revealed that bioinformatics tools specifically suited for protein sequence analysis and structure/function prediction may help in discriminating among existing and non-existing proteins (Tress et al., 2007, PNAS, 104:5495-5500). From this it is possible to speculate that the integration of different high performing tools, endowed with both low false positive and negative rates, is an efficient and fast way of annotating the spliced gene products resulting from predictive tools of alternative splicing. In Bologna, at the Biocomputing group a platform is under construction that will annotate all the products contained in ASPicDB, a data base of predicted spliced gene products at CASPUR (http://t.caspur.it/ASPicDB/).


The rice genome as a model for gene expression studies in plants

Molecular analysis of rice defence responses to blast infection

Pamela Abbruscato, Parco Tecnologico Padano

Plants developed a sophisticated machinery to perceive external signals and optimally respond to pathogens. A common facet of plant defence responses is the fine tuning of a large number of genes by transcriptional factors and the prompt expression of a battery of defence genes, such as pathogenesis related proteins (PR proteins). Among transcriptional factors, we characterized a panel of rice WRKY genes in response to different Magnaporthe grisea strains, to highlight differences between host and non-host resistance. We also analysed the expression of defence genes in selected Italian rice cultivars ranging from highly-susceptible to completely resistant, to investigate a potential correlation between the phenotype and the expression levels of the tested defence genes. Expression of thirty OsWRKY candidate genes was quantified by Real Time PCR after infection with host and non-host strains at 12, 24 and 48 hours post infection (hpi). Most of the up-regulated genes were found to be expressed in response to host infection and only a few by both host and non-host strains, whereas only two genes were induced specifically by non-host M. grisea strains. Then, we quantified the expression of the five defence genes before and after infection with two host Italian strains of M. grisea. We selected the chitinase class III and β-1,3-glucanase due to their well-recognised antifungal activity and peroxidase 22.3 given its role in reinforcing the cell wall. We also included PR1 and PBZ1 genes, as they are known markers of the activation of defence responses. In resistant cultivars we observed a strong induction of several defence genes at once after blast infection. In contrast, in the susceptible cultivars we did not observe any variation of expression, confirming the role of these defence genes in resistance to blast.