Linear motifs confer functional diversity onto splice variants

The pre-translational modification of messenger ribonucleic acids (mRNAs) by alternative promoter usage and alternative splicing is an important source of pleiotropy. Despite intensive efforts, our understanding of the functional implications of this dynamically created diversity is still incomplete. Using the available knowledge of interaction modules, particularly within intrinsically disordered regions (IDRs), we analysed the occurrences of protein modules within alternative exons. We find that regions removed or included by pre-translational variation are enriched in linear motifs suggesting that the removal or inclusion of exons containing these interaction modules is an important regulatory mechanism. In particular, we observe that PDZ-, PTB-, SH2- and WW-domain binding motifs are more likely to occur within alternative exons. We also determine that regions removed or included by alternative promoter usage are enriched in IDRs suggesting that protein isoform diversity is tightly coupled to the modulation of IDRs. This study, therefore, demonstrates that short linear motifs are key components for establishing protein diversity between splice variants.     

Distinct Types of Disorder in the Human Proteome: Functional Implications for Alternative Splicing

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as “flexible” and “constrained” conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer.     

Tissue-Specific Functional Networks for Prioritizing Phenotype and Disease Genes

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as “functionality” and “functional relationships” are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.     

Competition of PTB with TIA proteins for binding to a U-rich cis-element determines tissue-specific splicing of the myosin phosphatase targeting subunit 1

A considerable amount of smooth muscle phenotypic diversity is generated by tissue-specific and developmentally regulated splicing of alternative exons. The control mechanisms are unknown. We are using a myosin phosphatase targeting subunit-1 (MYPT1) alternative exon as a model to investigate this question. In the present study, we show that the RNA binding proteins TIA and PTB function as antagonistic enhancers and suppressors of splicing of the alternative exon, respectively. Each functions through a single U-rich element, containing two UCUU motifs, just downstream of the alternative exon 5' splice site. Tissue-specific down-regulation of TIA protein in the perinatal period allows PTB to bind to the U-rich element and suppress splicing of the alternative exon as the visceral smooth muscle acquires the fast-phasic smooth muscle contractile phenotype. This provides a novel role for PTB in the tissue-specific regulation of splicing of alternative exons during the generation of smooth muscle phenotypic diversity.     

Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation

Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA (“STAR” motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.     

Prediction of short linear protein binding regions

Short linear motifs in proteins (typically 3-12 residues in length) play key roles in protein-protein interactions by frequently binding specifically to peptide binding domains within interacting proteins. Their tendency to be found in disordered segments of proteins has meant that they have often been overlooked. Here we present SLiMPred (short linear motif predictor), the first general de novo method designed to computationally predict such regions in protein primary sequences independent of experimentally defined homologs and interactors. The method applies machine learning techniques to predict new motifs based on annotated instances from the Eukaryotic Linear Motif database, as well as structural, biophysical, and biochemical features derived from the protein primary sequence. We have integrated these data sources and benchmarked the predictive accuracy of the method, and found that it performs equivalently to a predictor of protein binding regions in disordered regions, in addition to having predictive power for other classes of motif sites such as polyproline II helix motifs and short linear motifs lying in ordered regions. It will be useful in predicting peptides involved in potential protein associations and will aid in the functional characterization of proteins, especially of proteins lacking experimental information on structures and interactions. We conclude that, despite the diversity of motif sequences and structures, SLiMPred is a valuable tool for prioritizing potential interaction motifs in proteins.     

Protein domains enriched in mammalian tissue-specific or widely expressed genes

In multicellular organisms some genes are expressed in essentially all tissues, whereas others are expressed predominantly in only one or a few tissues. In this study, we investigate the relationship between the tissue-specificity of gene expression and the type of protein encoded. We find that many protein domains are found to be enriched in either tissue-specific or widely expressed genes. Domains enriched in tissue-specific genes tend to be metazoan-specific; these same domains are also enriched in genes that are not essential for cell viability. These findings identify families of proteins that are probably used in the development or terminal differentiation of many different tissue types.     

Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing

All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-mRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.     

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.     

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.