Splicing Of mRNA Precursors Research Articles

Role of Precursor mRNA Splicing in Nutrient-Induced Alterations in Gene Expression and Metabolism1–3

Precursor mRNA (pre-mRNA) splicing is a critical step in gene expression that results in the removal of intronic sequences from immature mRNA, leading to the production of mature mRNA that can be translated into protein. Alternative pre-mRNA splicing is the process whereby alternative exons and/or introns are selectively included or excluded, generating mature mRNAs that encode proteins that may differ in function. The resulting alterations in the pattern of protein isoform expression can result in changes in protein–protein interaction, subcellular localization, and flux through metabolic pathways. Although basic mechanisms of pre-mRNA splicing of introns and exons are reasonably well characterized, how these mechanisms are regulated remains poorly understood. The goal of this review is to highlight selected recent advances in our understanding of the regulation of pre-mRNA splicing by nutrients and modulation of nutrient metabolism that result from changes in pre-mRNA splicing.

Global Promotion of Alternative Internal Exon Usage by mRNA 3′ End Formation Factors

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

Chronic HIV-1 Tat and HIV reduce Rbfox3/NeuN: evidence for sex-related effects.

The NeuN antibody has been widely used to identify and quantify neurons in normal and disease situations based on binding to a nuclear epitope in most types of neurons. This epitope was recently identified as the RNA-binding, feminizing locus on X-3 (Rbfox3), a member of the larger, mammalian Fox1 family of RNA binding proteins. Fox1 proteins recognize a unique UGCAUG mRNA motif and regulate alternative splicing of precursor mRNA to control post-transcriptional events important in neuronal differentiation and central nervous system development. Recent clinical findings show that Rbfox3/NeuN gene dosage is altered in certain human neurodevelopmental disorders, and redistribution has been noted in HIV(+) tissue. We hypothesized that HIV-1 Tat might affect Rbfox3/NeuN expression, and examined this question in vivo using inducible transgenic mice, and in vitro using human mesencephalic-derived neurons. Rbfox3/NeuN expression and localization in HIV+ basal ganglia and hippocampus was also examined. Chronic Tat exposure reduced Rbfox3/NeuN protein levels and increased cytoplasmic localization, similar to the effect of HIV exposure. Cytoplasmic Rbfox3/NeuN signal has occasionally been reported, although the meaning or function of cytoplasmic versus nuclear localization remains speculative. Importantly, Rbfox3/NeuN reductions were more significant in male mice. Although Rbfox3/NeuN-expressing cells were significantly decreased by Tat exposure, stereology showed that Nissl(+) neuron numbers remained normal. Thus, loss of Rbfox3/NeuN may relate more to functional change than to neuron loss. The effects of Tat by itself are highly relevant to HIV(+) individuals maintained on antiretroviral therapy, since Tat is released from infected cells even when viral replication is inhibited.

The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes.

Aquarius is a multifunctional putative RNA helicase that binds precursor-mRNA introns at a defined position. Here we report the crystal structure of human Aquarius, revealing a central RNA helicase core and several unique accessory domains, including an ARM-repeat domain. We show that Aquarius is integrated into spliceosomes as part of a pentameric intron-binding complex (IBC) that, together with the ARM domain, cross-links to U2 snRNP proteins within activated spliceosomes; this suggests that the latter aid in positioning Aquarius on the intron. Aquarius's ARM domain is essential for IBC formation, thus indicating that it has a key protein-protein-scaffolding role. Finally, we provide evidence that Aquarius is required for efficient precursor-mRNA splicing in vitro. Our findings highlight the remarkable structural adaptations of a helicase to achieve position-specific recruitment to a ribonucleoprotein complex and reveal a new building block of the human spliceosome.

Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

Mechanism of alternative splicing and its regulation.

Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development. Regulation of alternative splicing is a complicated process in which numerous interacting components are at work, including cis-acting elements and trans-acting factors, and is further guided by the functional coupling between transcription and splicing. Additional molecular features, such as chromatin structure, RNA structure and alternative transcription initiation or alternative transcription termination, collaborate with these basic components to generate the protein diversity due to alternative splicing. All these factors contributing to this one fundamental biological process add up to a mechanism that is critical to the proper functioning of cells. Any corruption of the process may lead to disruption of normal cellular function and the eventuality of disease. Cancer is one of those diseases, where alternative splicing may be the basis for the identification of novel diagnostic and prognostic biomarkers, as well as new strategies for therapy. Thus, an in-depth understanding of alternative splicing regulation has the potential not only to elucidate fundamental biological principles, but to provide solutions for various diseases.

Abstract 4277: Alternative splicing in cancer transcriptomes

Abstract Alternative splicing of precursor mRNA is a key mechanism of eukaryotic gene regulation. Cancer-associated changes in alternative splicing can modulate gene function or activity, and consequently affect downstream molecular pathways critical for tumorigenesis or metastasis. RNA-Seq has become a powerful technology for genome-wide analysis of alternative splicing. We have developed sensitive and robust computational methods for quantitative analysis of alternative splicing using RNA-Seq data. Using these methods, we have analyzed the alternative splicing profiles of >4000 cancer samples across multiple types of cancer. Our analysis has identified important alternative splicing events that may contribute to the development and progression of cancer. Note: This abstract was not presented at the meeting. Citation Format: Shihao Shen, Ying Nian Wu, Yi Xing. Alternative splicing in cancer transcriptomes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2014-4277

Uncovering global SUMOylation signaling networks in a site-specific manner.

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution mass spectrometry, we have studied global SUMOylation in human cells and in a site-specific manner, identifying a total of over 4,300 SUMOylation sites in over 1,600 proteins. Moreover, for the first time in excess of 1,000 SUMOylation sites were identified under standard growth conditions. SUMOylation dynamics were quantitatively studied in response to SUMO protease inhibition, proteasome inhibition and heat shock. A considerable amount of SUMOylated lysines have previously been reported to be ubiquitylated, acetylated or methylated, indicating crosstalk between SUMO and other post-translational modifications. We identified 70 phosphorylation and 4 acetylation events in close proximity to SUMOylation sites, and provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, pre-mRNA splicing and ribosome assembly.

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex.

The precursor mRNA (pre-mRNA) retention and splicing (RES) complex is a spliceosomal complex that is present in yeast and humans and is important for RNA splicing and retention of unspliced pre-mRNA. Here, we present the solution NMR structure of the RES core complex from Saccharomyces cerevisiae. Complex formation leads to an intricate folding of three components-Snu17p, Bud13p and Pml1p-that stabilizes the RNA-recognition motif (RRM) fold of Snu17p and increases binding affinity in tertiary interactions between the components by more than 100-fold compared to that in binary interactions. RES interacts with pre-mRNA within the spliceosome, and through the assembly of the RES core complex RNA binding efficiency is increased. The three-dimensional structure of the RES core complex highlights the importance of cooperative folding and binding in the functional organization of the spliceosome.

Single-molecule fluorescence-based studies on the dynamics, assembly and catalytic mechanism of the spliceosome.

Pre-mRNA (precursor mRNA) splicing is a key step in cellular gene expression where introns are excised and exons are ligated together to produce mature mRNA. This process is catalysed by the spliceosome, which consists of five snRNPs (small nuclear ribonucleoprotein particles) and numerous protein factors. Assembly of these snRNPs and associated proteins is a highly dynamic process, making it challenging to study the conformational rearrangements and spliceosome assembly kinetics in bulk studies. In the present review, we discuss recent studies utilizing techniques based on single-molecule detection that have helped overcome this challenge. These studies focus on the assembly dynamics and splicing kinetics in real-time, which help understanding of spliceosomal assembly and catalysis.

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis.

BackgroundAlternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress.ResultsTo analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS.ConclusionsOur study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-431) contains supplementary material, which is available to authorized users.

CFLIP expression is altered in severe corticosteroid-resistant asthma.

Dysregulation of alternative splicing of mRNA precursors is known to contribute to numerous human diseases. In this study we carried out the first systematic search for asthma-associated changes in alternative splicing events, using a model of Aspergillus fumigatus (A. fumigatus)-sensitized mice and an exon junction microarray to detect potential changes in alternative splicing. One of the sensitization-associated changes identified in the search was a shift in alternative splicing of the mRNA encoding cFLIP, a modulator of the caspase-mediated extrinsic apoptosis pathway. Expanding these studies to human asthma patients, we discovered a significant decrease in the expression of both cFLIP isoforms in severe corticosteroid-resistant asthmatics. Although it is unclear whether these changes were due solely to differences in alternative splicing, these findings provide evidence that dysregulation of the extrinsic apoptosis pathway is part of the underlying immunopathogenesis of severe refractory asthma.

Characterization of Rubisco Activase Genes in Maize: An α-Isoform Gene Functions alongside a β-Isoform Gene

Rubisco activase (RCA) catalyzes the activation of Rubisco in vivo and plays a crucial role in regulating plant growth. In maize (Zea mays), only β-form RCA genes have been cloned and characterized. In this study, a genome-wide survey revealed the presence of an α-form RCA gene and a β-form RCA gene in the maize genome, herein referred to as ZmRCAα and ZmRCAβ, respectively. An analysis of genomic DNA and complementary DNA sequences suggested that alternative splicing of the ZmRCAβ precursor mRNA (premRNA) at its 3' untranslated region could produce two distinctive ZmRCAβ transcripts. Analyses by electrophoresis and matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry showed that ZmRCAα and ZmRCAβ encode larger and smaller polypeptides of approximately 46 and 43 kD, respectively. Transcriptional analyses demonstrated that the expression levels of both ZmRCAα and ZmRCAβ were higher in leaves and during grain filling and that expression followed a specific cyclic day/night pattern. In 123 maize inbred lines with extensive genetic diversity, the transcript abundance and protein expression levels of these two RCA genes were positively correlated with grain yield. Additionally, both genes demonstrated a similar correlation with grain yield compared with three C₄ photosynthesis genes. Our data suggest that, in addition to the β-form RCA-encoding gene, the α-form RCA-encoding gene also contributes to the synthesis of RCA in maize and support the hypothesis that RCA genes may play an important role in determining maize productivity.

Glycosylation of Immunoglobulin G: Role of Genetic and Epigenetic Influences

ObjectiveTo determine the extent to which genetic and epigenetic factors contribute to variations in glycosylation of immunoglobulin G (IgG) in humans.Methods76 N-glycan traits in circulating IgG were analyzed by UPLC in 220 monozygotic and 310 dizygotic twin pairs from TwinsUK. A classical twin study design was used to derive the additive genetic, common and unique environmental components defining the variance in these traits. Epigenome-wide association analysis was performed using the Illumina 27k chip. Results51 of the 76 glycan traits studied have an additive genetic component (heritability, h 2)≥ 0.5. In contrast, 12 glycan traits had a low genetic contribution (h2<0.35). We then tested for association between methylation levels and glycan levels (P<2 x10-6). Among glycan traits with low heritability probe cg08392591 maps to a CpG island 5’ from the ANKRD11 gene, a p53 activator on chromosome 16. Probe cg26991199 maps to the SRSF10 gene involved in regulation of RNA splicing and particularly in regulation of splicing of mRNA precursors upon heat shock. Among those with high heritability we found cg13782134 (mapping to the NRN1L gene) and cg16029957 mapping near the QPCT gene to be array-wide significant. The proportion of array-wide epigenetic associations was significantly larger (P<0.005) among glycans with low heritability (42%) than in those with high heritability (6.2%).ConclusionsGlycome analyses might provide a useful integration of genetic and non-genetic factors to further our understanding of the role of glycosylation in both normal physiology and disease.

Post-transcriptional modifications modulate conformational dynamics in human U2–U6 snRNA complex

The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2-U6 complex in vitro. Consistent with yeast, the human U2-U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2-U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA-protein interactions in vivo.

Misregulation of Pre-mRNA Alternative Splicing in Cancer

Alternative splicing of mRNA precursors enables one gene to produce multiple protein isoforms with differing functions. Under normal conditions, this mechanism is tightly regulated in order for the human genome to generate proteomic diversity sufficient for the functional requirements of complex tissues. When deregulated, however, cancer cells take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Here, we discuss aspects of alternative splicing misregulation in cancer, focusing on splicing events affected by deregulation of regulatory splicing factors and also recent studies identifying mutated components of the splicing machinery. An increasing body of evidence indicates that aberrant splicing of mRNA precursors leads to production of aberrant proteins that contribute to tumorigenesis. Recent studies show that alterations in cellular concentrations of regulatory splicing factors and mutations in components of the core splicing machinery provide major mechanisms of misregulation of mRNA splicing in cancer. A better understanding of this misregulation will potentially reveal a group of novel drug targets for therapeutic intervention.

Complexity of the Alternative Splicing Landscape in Plants

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Genome-Wide Detection of Condition-Sensitive Alternative Splicing in Arabidopsis Roots

Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms.

Rapid screening of yeast mutants with reporters identifies new splicing phenotypes

Nuclear precursor mRNA splicing requires the stepwise assembly of a large complex, the spliceosome. Recent large-scale analyses, including purification of splicing complexes, high-throughput genetic screens and interactomic studies, have linked numerous factors to this dynamic process, including a well-defined core conserved from yeast to human. Intriguingly, despite extensive studies, no splicing defects were reported for some of the corresponding yeast mutants. To resolve this paradox, we screened a collection of viable yeast strains carrying mutations in splicing-related factors with a set of reporters including artificial constructs carrying competing splice sites. Previous analyses have indeed demonstrated that this strategy identifies yeast factors able to regulate alternative splicing and whose properties are conserved in human cells. The method, sensitive to subtle defects, revealed new splicing phenotypes for most analyzed factors such as the Urn1 protein. Interestingly, a mutant of PRP8 specifically lacking an N-terminal proline-rich region stimulated the splicing of a reporter containing competing branchpoint/3' splice site regions. Thus, using appropriate reporters, yeast can be used to quickly delineate the effect of various factors on splicing and identify those with the propensity to regulate alternative splicing events.

Phylogenetic and Molecular Characterization of the Splicing Factor RBM4

The mammalian multi-functional RNA-binding motif 4 (RBM4) protein regulates alterative splicing of precursor mRNAs and thereby affects pancreas and muscle cell differentiation. RBM4 homologs exist in all metazoan lineages. The C-terminal unstructured domain of RBM4 is evolutionarily divergent and contains stretches of low-complexity sequences, including single amino acid and/or dipeptide repeats. Here we examined the splicing activity, phosphorylation potential, and subcellular localization of RBM4 homologs from a wide range of species. The results show that these RBM4 homologs exert different effects on 5′ splice site utilization and exon selection, and exhibit different subnuclear localization patterns. Therefore, the C-terminal domain of RBM4 may contribute to functional divergence between homologs. On the other hand, analysis of chimeric human RBM4 proteins containing heterologous sequences at the C-terminus revealed that the N-terminal RNA binding domain of RBM4 could have a dominant role in determining splicing outcome. Finally, all RBM4 homologs examined could be phosphorylated by an SR protein kinase, suggesting that they are regulated by a conserved mechanism in different species. This study offers a first clue to functional evolution of a splicing factor.