Project description:Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.
Project description:The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.
Project description:Pre-mRNA splicing factors recognize consensus signals within preliminary transcripts, and as cogs of the spliceosome machine, orchestrate the excision and rejoining of pre-mRNA regions for gene expression. Large-scale sequencing has demonstrated that mutations in key genes encoding pre-mRNA splicing factors are common among myeloid neoplasms and also occur in a variety of other cancers. This revelation offers new therapeutic opportunities to target pre-mRNA splicing vulnerabilities in hematologic and other malignancies. The mutated residues typically alter 3' splice site choice for a subset of transcripts. In this review, we highlight mechanistic insights from recent 3D structures that reveal the affected residues poised for pre-mRNA recognition.
Project description:Whereas individual steps of protein-coding gene expression in eukaryotes can be studied in isolation in vitro, it has become clear that these steps are intimately connected within cells. Connections not only ensure quality control but also fine-tune the gene expression process, which must adapt to environmental changes while remaining robust. In this review, we systematically present proven and potential mechanisms by which sequence-specific DNA-binding transcription factors can alter gene expression beyond transcription initiation and regulate pre-mRNA splicing, and thereby mRNA isoform production, by (i) influencing transcription elongation rates, (ii) binding to pre-mRNA to recruit splicing factors, and/or (iii) blocking the association of splicing factors with pre-mRNA. We propose various mechanistic models throughout the review, in some cases without explicit supportive evidence, in hopes of providing fertile ground for future studies.
Project description:Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3' splice-site. PUF60 has homology to U2AF(65), a general splicing factor that facilitates 3' splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF(65)in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF(65) in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF(65) levels can dictate alternative splicing patterns. Our results indicate that recognition of 3' splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing.
Project description:Cotranscriptional recruitment of pre-mRNA splicing factors to their genomic targets facilitates efficient and ordered assembly of a mature messenger ribonucleoprotein particle (mRNP). However, how the cotranscriptional recruitment of splicing factors is regulated remains largely unknown. Here, we demonstrate that protein arginine methylation plays a novel role in regulating this process in Saccharomyces cerevisiae. Our data show that Hmt1, the major type I arginine methyltransferase, methylates Snp1, a U1 small nuclear RNP (snRNP)-specific protein, and that the mammalian Snp1 homolog, U1-70K, is likewise arginine methylated. Genome-wide localization analysis reveals that the deletion of the HMT1 gene deregulates the recruitment of U1 snRNP and its associated components to intron-containing genes (ICGs). In the same context, splicing factors acting downstream of U1 snRNP addition bind to a reduced number of ICGs. Quantitative measurement of the abundance of spliced target transcripts shows that these changes in recruitment result in an increase in the splicing efficiency of developmentally regulated mRNAs. We also show that in the absence of either Hmt1 or of its catalytic activity, an association between Snp1 and the SR-like protein Npl3 is substantially increased. Together, these data support a model whereby arginine methylation modulates dynamic associations between SR-like protein and pre-mRNA splicing factor to promote target specificity in splicing.
Project description:Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors (PRPF3, 4, 6, 8, 31, SNRNP200, and RP9) have been linked to 15-20% of autosomal dominant RP (adRP) cases. Current evidence indicates that PRPF mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis. Importantly, additional PRPF functions beyond RNA splicing have been documented recently, suggesting a more complex mechanism underlying PRPF-RPs driven disease pathogenesis. The current review focuses on the key RP-PRPF genes, depicting the current understanding of their roles in RNA splicing, impact of their mutations on retinal cell's transcriptome and phenome, discussed in the context of model species including yeast, zebrafish, and mice. Importantly, information on PRPF functions beyond RNA splicing are discussed, aiming at a holistic investigation of PRPF-RP pathogenesis. Finally, work performed in human patient-specific lab models and developing gene and cell-based replacement therapies for the treatment of PRPF-RPs are thoroughly discussed to allow the reader to get a deeper understanding of the disease mechanisms, which we believe will facilitate the establishment of novel and better therapeutic strategies for PRPF-RP patients.
Project description:As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.
Project description:Chromatin organization is highly dynamic and regulates transcription. Upon transcriptional activation, chromatin is remodeled and referred to as "open," but quantitative and dynamic data of this decompaction process are lacking. Here, we have developed a quantitative high resolution-microscopy assay in living yeast cells to visualize and quantify chromatin dynamics using the GAL7-10-1 locus as a model system. Upon transcriptional activation of these three clustered genes, we detect an increase of the mean distance across this locus by >100 nm. This decompaction is linked to active transcription but is not sensitive to the histone deacetylase inhibitor trichostatin A or to deletion of the histone acetyl transferase Gcn5. In contrast, the deletion of SNF2 (encoding the ATPase of the SWI/SNF chromatin remodeling complex) or the deactivation of the histone chaperone complex FACT lead to a strongly reduced decompaction without significant effects on transcriptional induction in FACT mutants. Our findings are consistent with nucleosome remodeling and eviction activities being major contributors to chromatin reorganization during transcription but also suggest that transcription can occur in the absence of detectable decompaction.
Project description:Recent cryo-EM structures of a group II intron caught in the process of invading DNA have given new insight into the mechanisms of both splicing and retrotransposition. Conformational dynamics involving the branch-site helix domain VI are responsible for substrate exchange between the two steps of splicing. These structural rearrangements have strong parallels with the movement of the branch-site helix in the spliceosome during catalysis. This is strong evidence for the spliceosome evolving from a group II intron ancestor. We observe other topological changes in the overall structure of the catalytic domain V that may occur in the spliceosome as well. Therefore, studying group II introns not only provides us with insight into the evolutionary origins of the spliceosome, but also may inform the design of experiments to further probe structure-function relationships in this eukaryotic splicing apparatus. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.