The relevance of alternative RNA splicing to pharmacogenomics

The importance of alternative RNA splicing in the generation of genetic diversity is now widely accepted. This article highlights how alternative RNA splicing can have an impact on drug efficacy and safety, and demonstrates its potential pharmacogenomic value. The analysis of the repertoire of alternative RNA splicing events could potentially identify markers of pharmacogenomic relevance with high sensitivity and specificity and also provides a route through which genes can be selected for single nucleotide polymorphism (SNP) genotyping. Recent methodological advances, including microarray and splice-dedicated expression profiling, have made it possible to perform high-throughput alternative splicing analyses.     

Cyclooxygenase variants: the role of alternative splicing

Alternative splicing of cellular pre-mRNA is responsible for production of multiple mRNAs from individual genes. Splice variants are expressed in cell- and tissue-specific contexts that are important in development and physiology. Alternative splicing can serve as a regulatory mechanism whereby developmental programming and environmental factors/stimuli affect biological activities of translated proteins. Cyclooxygenase (COX)-1 and -2 genes produce splice variants whose biological expression, relevance, and activities have been of significant interest. COX variants are produced by a variety of splicing mechanisms. Four structural domains of the COX proteins (the amino terminal signal peptide, membrane-binding domain, dimerization domain, and catalytic domain) are defined by specific COX exons. COX splice variants may, therefore, result in potential changes in protein subcellular localization, dimerization, and activity. COX variant proteins may act in roles which diverge from those of COX-1 and -2.     

Sex differentiation: the role of alternative splicing.

Sex differentiation in Drosophila is controlled by a regulatory cascade with at least three regulated alternative RNA-processing events. The results of recent work have verified much of the earlier molecular and genetic work in this field and have provided a demonstration that both positive and negative regulatory mechanisms are involved.     

Dynamic equilibria in protein kinases

Structural changes involved in protein kinase activation and ligand binding have been determined from a wealth of X-ray crystallographic evidence. Recent solution studies using NMR, EPR, HX-MS, and fluorescence techniques have deepened this understanding by highlighting the underlying energetics and dynamics of multistate conformational ensembles. This new research is showing how activation mechanisms and ligand binding alter the internal motions of kinases and enable allosteric coupling between distal regulatory regions and the active site.     

Post-transcriptional regulation of the creatine transporter gene: Functional relevance of alternative splicing

Background

Aberrations in about 10–15% of X-chromosome genes account for intellectual disability (ID); with a prevalence of 1–3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter (CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8–SLC6A8C, containing intron 4 and exons 5–13, was identified. Herein, we report the identification of a novel variant — SLC6A8D, and functional relevance of these isoforms.

Methods

Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport.

Results

SLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift). Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8, both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform resulted in a significant increase in creatine accumulation compared to CTR1 cells re-infected with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into HEK293 cells resulted in significantly higher creatine uptake.

Conclusions

CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake.

General significance

Provides added insight into the mechanism(s) of creatine transport regulation.     

Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.     

Mitogen-activated protein kinases in synaptic plasticity and memory

This review highlights five areas of recent discovery concerning the role of extracellular-signal regulated kinases (ERKs) in the hippocampus. First, ERKs have recently been directly implicated in human learning through studies of a human mental retardation syndrome. Second, new models are being formulated for how ERKs contribute to molecular information processing in dendrites. Third, a role of ERKs in stabilizing structural changes in dendritic spines has been defined. Fourth, a crucial role for ERKs in regulating local dendritic protein synthesis is emerging. Fifth, the importance of ERK interactions with scaffolding and structural proteins at the synapse is increasingly apparent. These topics are discussed within the context of an emerging role for ERKs in a wide variety of forms of synaptic plasticity and memory formation in the behaving animal.     

The role of alternative pre-mRNA splicing in regulating the structure and function of skeletal protein 4.1

Alternative pre-mRNA splicing plays a major role in regulating cell type-specific expression of the protein 4.1 family of skeletal proteins. The biological importance of alternative splicing as a mechanism for 4.1 gene regulation is underscored by studies of the prototypical 4.1R gene in erythroid cells: activation of exon 16 inclusion in mRna at the erythroblast stage greatly enhances the ability of newly synthesized 4.1R protein to bind spectrin and actin, and thus assemble into a stable membrane skeleton. This gain-of- function has profound effects on the biophysical properties of deformability and membrane strength that are critical to red cell survival in the circulation. Another example of developmentally regulated splicing occurs in differentiating mammary epithelial cells in culture, where cell morphogenesis is accompanied by a splicing switch that reversibly activates inclusion of alternative exon muscle. Few other genes are known to be so richly endowed with regulated switches in pre-mRna splicing making the 4.1R gene an interesting paradigm for the role of alternative splicing as a mediator of cell function. Recent evidence that other members of the 4.1 gene family are also regulated by alternative splicing suggests, moreover, that this phenomenon is of general importance in regulating the structure of this class of skeletal proteins.     

The emerging role of alternative splicing in senescence and aging

Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age‐related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF‐1, SIRT1, and ING‐1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype.     

Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing

In plants, calcium-dependent calmodulin-independent protein kinases (CDPKs) are the predominant calcium-regulated protein kinases and their genes are encoded by a multigene family. A CDPK gene was cloned from a liverwort, Marchantia polymorpha, which showed a high level of sequence similarities to other higher plant CDPK genes. The liverwort CDPK gene consisted of 9 exons and 8 introns. The 6th and 7th exons (Exon 6A and Exon 6B) were almost identical except for 4-amino acid substitutions, both of which coded for EF-hands in the calcium-binding domain. RT-PCR analysis revealed that two species of mature mRNA containing either Exon 6A or Exon 6B were generated from a single CDPK gene by mutually exclusive alternative splicing. Both histidine-tagged fusion proteins derived from cDNAs containing either Exon 6A or Exon 6B exhibited calcium-dependent protein kinase activity in vitro. Preferential accumulation of the mature mRNA with Exon 6A detected in male sexual organ implies possible sexual control of the ratio between the two CDPK isozymes through alternative splicing. Functions and evolution of CDPKs are discussed based on the structure and expression of the liverwort CDPK gene.     

Sudemycin E influences alternative splicing and changes chromatin modifications

Sudemycin E is an analog of the pre-messenger RNA splicing modulator {"type":"entrez-nucleotide","attrs":{"text":"FR901464","term_id":"525229801","term_text":"FR901464"}}FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes. Thus, in addition to the reversible changes in alternative splicing, sudemycin E causes changes in chromatin modifications that result in chromatin condensation, which is a likely contributing factor to cancer cell death.     

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.     

Regulation of alternative splicing by reversible protein phosphorylation

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.