The Alternative Choice of Constitutive Exons throughout Evolution

Alternative cassette exons are known to originate from two processes exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 59 splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicin

Base-specific mutational intolerance near splice sites clarifies the role of nonessential splice nucleotides

Variation in RNA splicing (i.e., alternative splicing) plays an important role in many diseases. Variants near 5' and 3' splice sites often affect splicing, but the effects of these variants on splicing and disease have not been fully characterized beyond the two "essential" splice nucleotides flanking each exon. Here we provide quantitative measurements of tolerance to mutational disruptions by position and reference allele-alternative allele combinations. We show that certain reference alleles are particularly sensitive to mutations, regardless of the alternative alleles into which they are mutated. Using public RNA-seq data, we demonstrate that individuals carrying such variants have significantly lower levels of the correctly spliced transcript, compared to individuals without them, and confirm that these specific substitutions are highly enriched for known Mendelian mutations. Our results propose a more refined definition of the "splice region" and offer a new way to prioritize and provide functional interpretation of variants identified in diagnostic sequencing and association studies.Peer reviewe

Assessment of orthologous splicing isoforms in human and mouse orthologous genes

<p>Abstract</p> <p>Background</p> <p>Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level.</p> <p>Results</p> <p>As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns.</p> <p>Conclusions</p> <p>We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level.</p

Role of alternative splicing in neurogenic commitment

To form complex organisms characterized by different tissues with specialized functions, cells must acquire distinct identities during development. Yet, all the cells of an organism are equipped with the same genomic information. Elucidating the mechanisms that regulate the determination of a cell identity, i.e. the cell-fate commitment, is a main purpose in developmental biology. Numerous studies focused on genes that are activated or repressed at each stage of differentiation, identifying several key regulators of development. However, this approach ignores the transcript variability derived from alternative splicing, the transcriptional process by which different gene coding segments, i.e. exons, are combined giving rise to multiple transcripts and proteins from the same gene. With the advent of novel sequencing technologies, it is becoming clear that alternative splicing is widespread in higher organisms, regulates several processes and presents tissue- and cell-specificity. In mammals, the brain shows the highest degree of alternative splicing, with neurons expressing a high variety of splice variants. In this project I investigated whether and how alternative splicing could regulate cell-fate determination in the context of the embryonic development of the mouse neocortex, a highly complex structure presenting several different neuronal subtypes generated at specific time points. For this purpose, I analyzed transcriptome data of cells of the neurogenic lineage isolated from the developing mouse neocortex at subsequent stages of differentiation. I showed that the expression pattern of the proteins regulating splicing, i.e. the splicing factors, changes during neocortical development. By employing several bioinformatic tools, I described the splicing profile that characterizes each differentiation stage and, for the first time, I identified the splicing events that mark cell-fate commitment to a neurogenic identity. Alternative splicing mostly involved genes with a role in nervous system development, cell growth and signaling, mainly leading to the production of alternative protein isoforms. Splicing choices taken during the neurogenic commitment were kept throughout neurogenesis. Thus, exons that start to be included during cell-fate determination are always included in post-mitotic neurons. Exons gained during neurogenic commitment were characterized by strong features in their upstream intron, presented a general short length with an overrepresentation of microexons in the 3-27 nucleotides length range and showed an enrichment for binding motifs of the neural splicing factor nSR100. In vivo manipulation in the embryonic mouse neocortex highlighted isoform-specific effects on neocortical development, strongly suggesting a causal relationship between alternative splicing choices and cell-fate commitment. Moreover, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed a better understanding of previously identified splicing events that characterize the nervous system and the relationships between neural-specific splicing factors.:Table of Contents Abstract I Zusammenfassung III Table of Contents V List of Figures VII List of Tables IX Abbreviations X Gene abbreviations XII 1 Introduction 1 1.1 Neurogenesis during embryonic development 2 1.1.1 Formation and patterning of the neural tube 2 1.1.2 Neural progenitors in the dorsal telencephalon 6 1.1.3 Neurogenesis 8 1.1.4 Regulation of neurogenesis 10 1.1.5 A novel tool to investigate cell-fate determination in the central nervous system: the Btg2RFP/Tubb3GFP mouse line 13 1.2 Alternative splicing: an additional level of genomic regulation 15 1.2.1 The splicing reaction 16 1.2.2 What makes splicing alternative? 18 1.2.3 Regulation of alternative splicing 19 1.2.4 The challenge to detect splicing 23 1.2.5 New sequencing technologies reveal a high transcriptome complexity 29 1.2.6 Splicing in nervous system development 31 1.2.7 Aims of the project 36 2 Materials and methods 38 2.1 Materials 38 2.1.1 Bacteria, cells, mouse strains 38 2.1.2 Vector 38 2.1.3 Primers 38 2.1.4 Chemicals and buffers 41 2.1.5 Antibodies 42 2.1.6 Kits and enzymes 42 2.2 Methods 43 2.2.1 Animal experiments 43 2.2.2 Molecular biology 44 2.2.3 Immunohistochemistry 46 2.2.4 Bioinformatics 47 3 Results 53 3.1 Splicing factors are differentially expressed during neurogenic commitment and neurogenesis 53 3.2 Detection of alternative splicing 55 3.2.1 Isoform-switching 55 3.2.2 Exon usage and splicing events 57 3.3 Validation 62 3.3.1 The isoform switching method has a poor validation rate 62 3.3.2 Analysis at the exon level has a high rate of validation 65 3.4 Pattern and representation of splicing events 67 3.4.1 Splicing choices during neurogenic commitment define the splicing profiles of neurons 67 3.4.2 Splicing events: microexon inclusion characterizes neurogenic commitment 69 3.5 Alternative splicing changes the protein output of genes involved in neurogenesis 75 3.5.1 Spliced genes are involved in neurogenesis and signaling 75 3.5.2 Impact of alternative splicing on the proteome 77 3.6 Splicing regulation: neural exon features and splicing factor binding 79 3.6.1 Included neural exons are short and preceded by strong exon-definition features 79 3.6.2 Early included exons are enriched for nSR100 binding sites 85 3.7 The Btg2RFP/Tubb3GFP mouse line outperforms previous models for the study of cell-type-specific splicing in the brain 88 3.8 In vivo manipulation of splice variants 90 4 Discussion 94 4.1 The combination of different bioinformatic approaches allows an accurate identification of splicing events at the exon-level 95 4.2 Splicing choices during neurogenic commitment establish a neural signature characterized by microexon inclusion 97 4.3 Splicing during neocortical development leads to the generation of alternative protein isoforms in genes involved in neurogenesis and signaling 98 4.4 Neural exons are short and present strong features facilitating inclusion 101 4.5 Neural exons are prevalently regulated by nSR100 during neurogenic commitment 102 4.6 In vivo overexpression of splice variants highlights isoform-specific functions in neurogenic commitment 105 4.7 Concluding remarks and future perspectives 108 5 Supplementary figures 110 6 References 118 Acknowledgments 137 Anlange I 138 Anlange II 13

Thermo-Sensitive Alternative Splicing of FLOWERING LOCUS M Is Modulated by Cyclin-Dependent Kinase G2

The ability to sense environmental temperature and to coordinate growth and development accordingly, is critical to the reproductive success of plants. Flowering time is regulated at the level of gene expression by a complex network of factors that integrate environmental and developmental cues. One of the main players, involved in modulating flowering time in response to changes in ambient temperature is FLOWERING LOCUS M (FLM). FLM transcripts can undergo extensive alternative splicing producing multiple variants, of which FLM-β and FLM-δ are the most representative. While FLM-β codes for the flowering repressor FLM protein, translation of FLM-δ has the opposite effect on flowering. Here we show that the cyclin-dependent kinase G2 (CDKG2), together with its cognate cyclin, CYCLYN L1 (CYCL1) affects the alternative splicing of FLM, balancing the levels of FLM-β and FLM-δ across the ambient temperature range. In the absence of the CDKG2/CYCL1 complex, FLM-β expression is reduced while FLM-δ is increased in a temperature dependent manner and these changes are associated with an early flowering phenotype in the cdkg2 mutant lines. In addition, we found that transcript variants retaining the full FLM intron 1 are sequestered in the cell nucleus. Strikingly, FLM intron 1 splicing is also regulated by CDKG2/CYCL1. Our results provide evidence that temperature and CDKs regulate the alternative splicing of FLM, contributing to flowering time definition

Usefulness and limitations of comprehensive characterization of mRNA splicing profiles in the definition of the clinical relevance of BRCA1/2 variants of uncertain significance

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses

Intronic motif pairs cooperate across exons to promote pre-mRNA splicing

A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition

Refining orthologue groups at the transcript level

<p>Abstract</p> <p>Background</p> <p>Orthologues are genes in different species that are related through divergent evolution from a common ancestor and are expected to have similar functions. Many databases have been created to describe orthologous genes based on existing sequence data. However, alternative splicing (in eukaryotes) is usually disregarded in the determination of orthologue groups and the functional consequences of alternative splicing have not been considered. Most multi-exon genes can encode multiple protein isoforms which often have different functions and can be disease-related. Extending the definition of orthologue groups to take account of alternate splicing and the functional differences it causes requires further examination.</p> <p>Results</p> <p>A subset of the orthologous gene groups between human and mouse was selected from the InParanoid database for this study. Each orthologue group was divided into sub-clusters, at the transcript level, using a method based on the sequence similarity of the isoforms. Transcript based sub-clusters were verified by functional signatures of the cluster members in the InterPro database. Functional similarity was higher within than between transcript-based sub-clusters of a defined orthologous group. In certain cases, cancer-related isoforms of a gene could be distinguished from other isoforms of the gene. Predictions of intrinsic disorder in protein regions were also correlated with the isoform sub-clusters within an orthologue group.</p> <p>Conclusions</p> <p>Sub-clustering of orthologue groups at the transcript level is an important step to more accurately define functionally equivalent orthologue groups. This work appears to be the first effort to refine orthologous groupings of genes based on the consequences of alternative splicing on function. Further investigation and refinement of the methodology to classify and verify isoform sub-clusters is needed, particularly to extend the technique to more distantly related species.</p

Refining orthologue groups at the transcript level

<p>Abstract</p> <p>Background</p> <p>Orthologues are genes in different species that are related through divergent evolution from a common ancestor and are expected to have similar functions. Many databases have been created to describe orthologous genes based on existing sequence data. However, alternative splicing (in eukaryotes) is usually disregarded in the determination of orthologue groups and the functional consequences of alternative splicing have not been considered. Most multi-exon genes can encode multiple protein isoforms which often have different functions and can be disease-related. Extending the definition of orthologue groups to take account of alternate splicing and the functional differences it causes requires further examination.</p> <p>Results</p> <p>A subset of the orthologous gene groups between human and mouse was selected from the InParanoid database for this study. Each orthologue group was divided into sub-clusters, at the transcript level, using a method based on the sequence similarity of the isoforms. Transcript based sub-clusters were verified by functional signatures of the cluster members in the InterPro database. Functional similarity was higher within than between transcript-based sub-clusters of a defined orthologous group. In certain cases, cancer-related isoforms of a gene could be distinguished from other isoforms of the gene. Predictions of intrinsic disorder in protein regions were also correlated with the isoform sub-clusters within an orthologue group.</p> <p>Conclusions</p> <p>Sub-clustering of orthologue groups at the transcript level is an important step to more accurately define functionally equivalent orthologue groups. This work appears to be the first effort to refine orthologous groupings of genes based on the consequences of alternative splicing on function. Further investigation and refinement of the methodology to classify and verify isoform sub-clusters is needed, particularly to extend the technique to more distantly related species.</p