Unusual Intron Conservation near Tissue-Regulated Exons Found by Splicing Microarrays

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5′ splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families

Evolutionary impact of limited splicing fidelity in mammalian genes

The functional significance of most alternative splicing (AS) events, especially frame-shifting ones, has been controversial. Using human-mouse comparison, we demonstrate that frame-preserving AS events adapt and get fixed more rapidly than frame-shifting AS events; selection for smaller exon size is stronger in frame-preserving exons than in frame-shifting ones. These results suggest AS events introducing mild changes are generally favored during evolution and explain the excess of shorter, frame-preserving cassette exons in present mammalian genomes

Muscleblind-Like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTGexp) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSALR transgenic mouse model which expresses a pathogenic range CTGexp. In the present study, we addressed the possibility that MBNL1 sequestration by CUGexp RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1ΔE3/ΔE3 knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1ΔE3/ΔE3 knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1ΔE3/ΔE3 brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUGexp RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain

Functional analysis of DcpS as a candidate cutaneous squamous cell carcinoma susceptibility gene

DCPS is a candidate cutaneous squamous cell carcinoma (SCC) susceptibility gene as determined by allelic imbalance mapping of paired SCC and genomic blood DNA samples. DCPS shows no protein expression in 23% of human SCCs on a tissue microarray, and reduced protein expression in another 30%. This is in contrast to strong staining for DCPS in 100% of normal tissue samples. DCPS, a decapping scavenger enzyme, influences the pool of available cap-binding proteins and, in turn, impacts aspects of mRNA metabolism like pre-mRNA splicing and decay. The hypothesis driving this research is that DCPS acts as a tumor suppressor. To test this hypothesis, functional effects of increasing and decreasing expression of DcpS in a mouse keratinocyte cell line have been studied. First intron splicing and exon skipping is enhanced in DcpS overexpression cell lines, while splicing of the second intron is not affected by DcpS expression. DcpS knockdown cell lines were found to have more stable mRNA compared to a control cell line. DcpS knockdown cell lines exhibit less growth than normal and overexpression cell lines. Cell migration is not affected by DcpS expression. DcpS knockdown cell lines exhibit more apoptosis than normal and overexpression cell lines. Finally, there is a greater percentage of cells in the S and G2-M phases of the cell cycle in DcpS knockdown cell lines compared to mock and overexpression cell lines. From these studies it appears that while DcpS may affect mRNA splicing and stability, decreased levels of DcpS do not result in a cancer phenotype. Rather, decreased levels of DcpS seem to lead to an anti-tumorigenic phenotype. Future directions may include examining the effect of UV radiation on these phenotypes and determining other proteins with which DcpS interacts.Pelotonia Undergraduate Research FellowshipSchool of Allied Medical Professions ScholarshipA one-year embargo was granted for this item

A network of conserved co-occurring motifs for the regulation of alternative splicing

Cis-acting short sequence motifs play important roles in alternative splicing. It is now possible to identify such sequence motifs as conserved sequence patterns in genome sequence alignments. Here, we report the systematic search for motifs in the neighboring introns of alternatively spliced exons by using comparative analysis of mammalian genome alignments. We identified 11 conserved sequence motifs that might be involved in the regulation of alternative splicing. These motifs are not only significantly overrepresented near alternatively spliced exons, but they also co-occur with each other, thus, forming a network of cis-elements, likely to be the basis for context-dependent regulation. Based on this finding, we applied the motif co-occurrence to predict alternatively skipped exons. We verified exon skipping in 29 cases out of 118 predictions (25%) by EST and mRNA sequences in the databases. For the predictions not verified by the database sequences, we confirmed exon skipping in 10 additional cases by using both RT–PCR experiments and the publicly available RNA-Seq data. These results indicate that even more alternative splicing events will be found with the progress of large-scale and high-throughput analyses for various tissue samples and developmental stages

Au Family Short Retroposons Contribute to Transcriptional and Phenotypic Diversity in Tomato (Solanaceae)

Here we report the current gene impact of the Au family of SINEs in tomato. The genome of Solanum lycopersicum ‘Heinz 1706‘ SL3.0 -NCBI annotation release 103- was reference searched and the Au profile was characterized in-depth. Tomato genome comprises ca. 670 Au copies, of entire length -18.5%-or truncated, randomly inserted and eroded, forming three well supported (>80%) super clusters which disperse along the 12 chromosomes mirroring the subtelomeric gene distribution bias of the species. In tomato, the Au clade is largely localized at protein coding genes -69.5% introns, 7.8% 3UTRs, 2.1% 5UTRs, 1.2% CDSs- followed by genomic copies -18.3%-, long non coding RNA genes -1.4%- and pseudogenes -0.8%-. The 419 tomato genes harboring intronic Au are diverse, weakly associated considering biological processes and molecular functions, but include important traits such as stress response, hormone response or phenotype plasticity. Au was found to be transcribed inside circular RNAs derived from 12 genic loci. Exonic Au affect the transcriptional and/or translational profiles of 67 tomato genes, including biological/agronomical important ones, contributing to UTR length and composition, UTR transcript variants, CDS boundary definitions, protein domains and variants. We propose that biased survival of Au in tomato genes is an adaptive feature.Fil: Grabiele, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; ArgentinaFil: Aguilera, Patricia Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentin

Analysis of splicing patterns by pyrosequencing

Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT–PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3′ terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources

Dynamic usage of alternative splicing exons during mouse retina development

Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements