pre-mRNA Molecules Research Articles

Alternative Splicing in Plant Genes: A Means of Regulating the Environmental Fitness of Plants.

Gene expression can be regulated through transcriptional and post-transcriptional mechanisms. Transcription in eukaryotes produces pre-mRNA molecules, which are processed and spliced post-transcriptionally to create translatable mRNAs. More than one mRNA may be produced from a single pre-mRNA by alternative splicing (AS); thus, AS serves to diversify an organism’s transcriptome and proteome. Previous studies of gene expression in plants have focused on the role of transcriptional regulation in response to environmental changes. However, recent data suggest that post-transcriptional regulation, especially AS, is necessary for plants to adapt to a changing environment. In this review, we summarize recent advances in our understanding of AS during plant development in response to environmental changes. We suggest that alternative gene splicing is a novel means of regulating the environmental fitness of plants.

RNA splicing in human disease and in the clinic.

Defects at the level of the pre-mRNA splicing process represent a major cause of human disease. Approximately 15-50% of all human disease mutations have been shown to alter functioning of basic and auxiliary splicing elements. These elements are required to ensure proper processing of pre-mRNA splicing molecules, with their disruption leading to misprocessing of the pre-mRNA molecule and disease. The splicing process is a complex process, with much still to be uncovered before we are able to accurately predict whether a reported genomic sequence variant (GV) represents a splicing-associated disease mutation or a harmless polymorphism. Furthermore, even when a mutation is correctly identified as affecting the splicing process, there still remains the difficulty of providing an exact evaluation of the potential impact on disease onset, severity and duration. In this review, we provide a brief overview of splicing diagnostic methodologies, from in silico bioinformatics approaches to wet lab invitro and invivo systems to evaluate splicing efficiencies. In particular, we provide an overview of how the latest developments in high-throughput sequencing can be applied to the clinic, and are already changing clinical approaches.

Independent Stochastic Burst-Like Transcription of Mutant and Wildtype Alleles as Mechanism for Cell-to-Cell Functional Imbalance in Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy (HCM) is an inherited disease mainly linked to mutations in genes coding for sarcomeric proteins. Most of these mutations result in a similar phenotype which is characterized by hypertrophy, cellular and myofibrillar disarray and interstitial fibrosis within the myocardium. The similar phenotype suggests an as yet unknown trigger for HCM-development common to different mutations.In our previous work on HCM-mutations in MYH7 we found a large cell-to-cell functional variability in HCM-tissue samples, presumably due to cell-to-cell variability in the expression of mutant protein. This was supported by a large cell-to-cell variability in mutant mRNA. We hypothesized that this observed functional imbalance among individual cardiomyocytes in the cellular network of the myocardium results in hypertrophy, disarray and interstitial fibrosis, i.e., triggers development of the HCM phenotype. Furthermore, FISH-based visualization of active transcription sites showed a substantial proportion of nuclei without active transcription, indicating discontinuous, burst-like transcription of the MYH7-alleles.To test whether independent, stochastic, burst-like transcription of the mutant and wildtype MYH7-alleles could indeed account for all these observations, we programmed a numerical model where the transcription of both alleles is switched on and off stochastically, resulting in sequential generation of pre-mRNA, mRNA and myosin molecules with defined rate constants. Rate constants for generation and decay of pre-mRNA, mRNA and myosin were taken from the literature. The model shows that independent, stochastic, burst-like transcription can indeed account for all of our above mentioned experimental results by adjusting the on and off rates of the transcription yielding the large cell-to-cell variability in mutant mRNA and protein. The fraction of nuclei without active transcription sites is determined by the on/off rates of transcription and modulated by the pre-mRNA to mRNA transition.

Pharmacological inhibition of the spliceosome subunit SF3b triggers exon junction complex-independent nonsense-mediated decay

Spliceostatin A, meayamycin, and pladienolide B are small molecules that target the SF3b subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP). These compounds are attracting much attention as tools to manipulate splicing and for use as potential anti-cancer drugs. We investigated the effects of these inhibitors on mRNA transport and stability in human cells. Upon splicing inhibition, unspliced pre-mRNAs accumulated in the nucleus, particularly within enlarged nuclear speckles. However, a small fraction of the pre-mRNA molecules were exported to the cytoplasm. We identified the export adaptor ALYREF as being associated with intron-containing transcripts and show its requirement for the nucleo-cytoplasmic transport of unspliced pre-mRNA. In contrast, the exon junction complex (EJC) core protein eIF4AIII failed to form a stable complex with intron-containing transcripts. Despite the absence of EJC, unspliced transcripts in the cytoplasm were degraded by nonsense-mediated decay (NMD), suggesting that unspliced transcripts are degraded by an EJC-independent NMD pathway. Collectively, our results indicate that although blocking the function of SF3b elicits a massive accumulation of unspliced pre-mRNAs in the nucleus, intron-containing transcripts can still bind the ALYREF export factor and be transported to the cytoplasm, where they trigger an alternative NMD pathway.

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

Mutations in spliceosomal proteins and retina degeneration

ABSTRACTA majority of human genes contain non-coding intervening sequences – introns that must be precisely excised from the pre-mRNA molecule. This event requires the coordinated action of five major small nuclear ribonucleoprotein particles (snRNPs) along with additional non-snRNP splicing proteins. Introns must be removed with nucleotidal precision, since even a single nucleotide mistake would result in a reading frame shift and production of a non-functional protein. Numerous human inherited diseases are caused by mutations that affect splicing, including mutations in proteins which are directly involved in splicing catalysis. One of the most common hereditary diseases associated with mutations in core splicing proteins is retinitis pigmentosa (RP). So far, mutations in more than 70 genes have been connected to RP. While the majority of mutated genes are expressed specifically in the retina, eight target genes encode for ubiquitous core snRNP proteins (Prpf3, Prpf4, Prpf6, Prpf8, Prpf31, and SNRNP200/Brr2) and splicing factors (RP9 and DHX38). Why mutations in spliceosomal proteins, which are essential in nearly every cell in the body, causes a disease that displays such a tissue-specific phenotype is currently a mystery. In this review, we recapitulate snRNP functions, summarize the missense mutations which are found in spliceosomal proteins as well as their impact on protein functions and discuss specific models which may explain why the retina is sensitive to these mutations.

Evolutionary Insights into RNA trans-Splicing in Vertebrates.

Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint.

STaQTool: Spot tracking and quantification tool for monitoring splicing of single pre-mRNA molecules in living cells

The vast majority of human protein-coding genes contain up to 90% of non-coding sequence in the form of introns that must be removed from the primary transcripts or pre-mRNAs. Diverse forms of mRNAs encoded from a single gene are created by the differential use of splice sites and alternative splicing is rapidly evolving. Although the kinetic properties of splicing are thought to be critical for proofreading and regulatory mechanisms, tools for making direct experimental measurements of splicing rates are still limited. We recently developed a strategy that permits real-time imaging of fluorescent-labelled introns in single pre-mRNA molecules. Here we describe the software tool that we created for automatic tracking and quantification of intronic fluorescence at the site of transcription in live human cells.

Single-Molecule Imaging of RNA in Live Cells

Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Previous studies have imaged RNAs in living cells by genetically inserting the binding sites for bacteriophage coat proteins in the RNA of interest. However, multiple nascent RNAs were simultaneously detected at the site of transcription, necessitating a modelling approach to infer kinetic information. To circumvent these significant limitations and potential problems in data interpretation, we developed a strategy that permits direct tracking of single nascent pre-mRNA molecules in live cells. We are using this approach to study how kinetic mechanisms impact on RNA biogenesis.

The cap-binding site of influenza virus protein PB2 as a drug target

The RNA polymerase of influenza virus consists of three subunits: PA, PB1 and PB2. It uses a unique `cap-snatching' mechanism for the transcription of viral mRNAs. The cap-binding domain of the PB2 subunit (PB2cap) in the viral polymerase binds the cap of a host pre-mRNA molecule, while the endonuclease of the PA subunit cleaves the RNA 10-13 nucleotides downstream from the cap. The capped RNA fragment is then used as the primer for viral mRNA transcription. The structure of PB2cap from influenza virus H1N1 A/California/07/2009 and of its complex with the cap analog m(7)GTP were solved at high resolution. Structural changes are observed in the cap-binding site of this new pandemic influenza virus strain, especially the hydrophobic interactions between the ligand and the target protein. m(7)GTP binds deeper in the pocket than some other virus strains, much deeper than the host cap-binding proteins. Analysis of the new H1N1 structures and comparisons with other structures provide new insights into the design of small-molecule inhibitors that will be effective against multiple strains of both type A and type B influenza viruses.

Interactions of the TREX-2 complex with mRNP particle of β-tubulin 56D gene

mRNA transport from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. A pre-mRNA molecule undergoes modification, such as 5'-capping, splicing, and 3'-end processing, in the nucleus. The molecule being modified interacts with a large number of proteins and, thus, mRNP particles are formed. The binding of factors involved in nuclear export also occurs during transcription and mRNA processing. We have shown that the functioning of TREX-2, an mRNA export complex, is restricted to the nucleus. We used the method of RNA coprecipitation that enables the selective extraction of RNA-protein complexes from samples to show that the transcription elongation complex TREX interacts with mRNA of the β-tubulin 56D gene over the entire length of the molecule. The capping protein Cbp80 reacted both with the cap structure and with a specific part of the coding mRNA of the β-tubulin 56D gene. The TREX-2 complex that mediates mRNA export from the nucleus to the cytoplasm is bound to the same part of the coding sequence. Thus, we identified a common binding site for all of the complexes under investigation on the mRNA of β-tubulin 56D. Co-immunoprecipitation reactions performed with S2 cell extracts revealed interactions between the components of complexes involved in transcription elongation, maturation, and export of mRNA. The model of molecular folding for the mRNP particle involving the mRNA of β-tubulin 56D has been proposed.

Single-Molecule Live-Cell Visualization of Pre-mRNA Splicing.

Microscopy protocols that allow live-cell imaging of molecules and subcellular components tagged with fluorescent conjugates are indispensable in modern biological research. A breakthrough was recently introduced by the development of genetically encoded fluorescent tags that combined with fluorescence-based microscopic approaches of increasingly higher spatial and temporal resolution made it possible to detect single protein and nucleic acid molecules inside living cells. Here, we describe an approach to visualize single nascent pre-mRNA molecules and to measure in real time the dynamics of intron synthesis and excision.

Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease.

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.

Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus.

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by homozygous expansion of the guanine-adenine-adenine (GAA) repeats in intron 1 of the FXN gene leading to transcriptional repression of frataxin expression. Post-translational histone modifications that typify heterochromatin are enriched in the vicinity of the repeats, whereas active chromatin marks in this region are underrepresented in FRDA samples. Yet, the immediate effect of the expanded repeats on transcription progression through FXN and their long-range effect on the surrounding genomic context are two critical questions that remain unanswered in the molecular pathogenesis of FRDA. To address these questions, we conducted next-generation RNA sequencing of a large cohort of FRDA and control primary fibroblasts. This comprehensive analysis revealed that the GAA-induced silencing effect does not influence expression of neighboring genes upstream or downstream of FXN. Furthermore, no long-range silencing effects were detected across a large portion of chromosome 9. Additionally, results of chromatin immunoprecipitation studies confirmed that histone modifications associated with repressed transcription are confined to the FXN locus. Finally, deep sequencing of FXN pre-mRNA molecules revealed a pronounced defect in the transcription elongation rate in FRDA cells when compared with controls. These results indicate that approaches aimed to reactivate frataxin expression should simultaneously address deficits in transcription initiation and elongation at the FXN locus.

A Model for the Origin of the First mRNAs.

I present a model for the evolution of the genetic code that seems to predict, in a totally natural way, the origin of the first mRNAs. In particular, the model--bestowing to peptidated-RNAs the major catalytic role in the phase that triggered the genetic code origin--suggests that interactions between peptidated-RNAs led to the synthesis of these ancestral catalysts. Within every group of these interactions, a pre-mRNA molecule evolved that was able to direct all interactions between peptidated-RNAs of that particular group. This represented an improvement in the coding of these interactions compared to the interaction groups that did not evolve these pre-mRNAs. This would represent a natural and intrinsic tendency. Therefore, these molecules of pre-mRNAs were positively selected because they improved the synthesis of the catalysts through this first form of coding of interactions among peptidated-RNAs. Thus, according to the model were the pairings--involving a base number greater than three (ennuplet code)-between peptidated-RNAs and pre-mRNAs that would represent the first form of the genetic code. The evolution of this ennuplet code to the triplet code might have been simply triggered by the natural tendency to make the reading module-that is the interactions between peptidated-RNAs and pre-mRNAs--of the different ennuplets to the triplet uniform, because in this way the heterogeneity existing in interactions between the aminoacylated or peptidated-RNAs and pre-mRNAs was eliminated. That is to say, there might have been the natural tendency toward the triplets because these would have made these interactions more efficient, given that the ennuplets were at least more cumbersome and therefore less economic and with an inferior adaptive value; and also because the triplets would represent the simpler choice among that available given that the doublets would have codified too few meanings and quartets instead too many. Therefore, the genetic code would result from a very long era of interactions among peptidated-RNAs under the continuous and fundamental selective pressure for improving catalysts' syntheses and thus catalysis. The model is strongly corroborated by the explanation that the tmRNA molecule (transfer-messenger RNA) would seem to be the very molecule of pre-mRNAs that the model predicts. In other words, the tmRNA would be the molecular fossil of the evolutionary stages that led to the appearance of the first mRNAs.

Single-Molecule Imaging of RNA Splicing in Live Cells.

Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Here, we describe a strategy that permits direct measurements of intron dynamics in single pre-mRNA molecules in live cells. This approach reveals that splicing can occur much faster than previously proposed and opens new avenues for studying how kinetic mechanisms impact on RNA biogenesis.

Single-Molecule Imaging of Pre-mRNA Splicing

We have developed a single-molecule RNA imaging method to study the intracellular sites of splicing. In this approach introns and exons in natural genes are probed in situ with distinctively labeled sets of about 50 oligonucleotide probes. The attachment of so many probes renders each target molecule so intensely fluorescent that it becomes visible as a bright diffraction-limited spot. Spots that fluoresce in just one color are either free introns or spliced mRNAs, and those that are visible in both colors correspond to pre-mRNA molecules. Our imaging studies confirm that constitutively spliced introns are removed at the gene locus during transcription. However, during alternative splicing events regulated by RNA binding proteins Sex lethal (in fruit flies) and polypyrimidine tract binding protein (in HeLa cells), which ensure that only one splice form is produced in a particular cell type, splicing gets uncoupled from transcription and occurs after the release of pre-mRNA from the gene locus. Similar uncoupling occurrs when intronic polypyrimidine tracts are masked within hairpins. Live cell imaging of the post-transcriptional splicing events suggests that they occur at the periphery of nuclear speckles where many splicing factors and poly adenylated transcripts are sequestered. Recent genome-wide studies reveal that a fraction of alternative splicing events likely occur via this route.

Splicing of designer exons informs a biophysical model for exon definition.

Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3′ end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.

Identifying splicing regulatory elements with de Bruijn graphs.

Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation.

Development of therapeutic splice-switching oligonucleotides.

Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case.