Biology subjects

Gooding, C.

Publications and source records attributed to Gooding, C..

6 recordsLinked to original sources

bioRxiv2023

Mechanism of an alternative splicing switch mediated by cell-specific and general splicing regulators

Alternative pre-mRNA splicing is regulated by RNA binding proteins (RBPs) that activate or repress regulated splice sites. Repressive RBPs bind stably to target RNAs via multivalent interactions, which can be achieved by both homo-oligomerization and by interactions with other RBPs mediated by intrinsically disordered regions (IDRs). Cell-specific splicing decisions commonly involve the action of widely expressed RBPs that can bind around target exons, but without effect in the absence of a key cell-specific regulator. To address how cell-specific regulators collaborate with constitutive RBPs in alternative splicing regulation we used the smooth-muscle specific regulator RBPMS. Recombinant RBPMS is sufficient to switch cell specific alternative splicing of Tpm1 exon 3 in cell free assays by remodelling ribonucleprotein complexes and preventing assembly of ATP-dependent splicing complexes. This activity depends upon its C-terminal IDR, which facilitates dynamic higher-order self-assembly, cooperative binding to multivalent RNA, and interactions with other splicing co-regulators, including MBNL1 and RBFOX2. Our data show how a cell-specific RBP can co-opt more widely expressed regulatory RBPs to facilitate cooperative assembly of stable cell-specific regulatory complexes.

molecular biology↗

bioRxiv2022

Phosphorylation of the Smooth Muscle Master Splicing Regulator RBPMS Regulates its Splicing Activity

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

molecular biology↗

bioRxiv2022

Polypyrimidine Tract Binding Protein 1 regulates the activation of mouse CD8 T cells

We show that the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1) is dispensable for the development of naive mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen-specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naive CD8 T cell leading to IL-2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naive CD8 T cells in addition to regulating the abundance of [~]200 mRNAs. PTBP1 is required for the nuclear accumulation of c-Fos, NFATc2 and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1-promoted expression of the shorter A{beta}1 isoform and exon 13 skipped A{beta}2 isoform of the catalytic A-subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T cell activation.

immunology↗

bioRxiv2019

Identification of RBPMS as a smooth muscle master splicing regulator via proximity of its gene with super-enhancers

Alternative splicing (AS) programs are primarily controlled by regulatory RNA binding proteins (RBPs). It has been proposed that a small number of master splicing regulators might control cell-specific splicing networks and that these RBPs could be identified by proximity of their genes to transcriptional super-enhancers. Using this approach we identified RBPMS as a critical splicing regulator in differentiated vascular smooth muscle cells (SMCs). RBPMS is highly down-regulated during phenotypic switching of SMCs from a contractile to a motile and proliferative phenotype and is responsible for 20% of the AS changes during this transition. RBPMS directly regulates AS of numerous components of the actin cytoskeleton and focal adhesion machineries whose activity is critical for SMC function in both phenotypes. RBPMS also regulates splicing of other splicing, post-transcriptional and transcription regulators including the key SMC transcription factor Myocardin, thereby matching many of the criteria of a master regulator of AS in SMCs.

molecular biology↗

bioRxiv2018

Lamin A/C controls nuclear matrin-3 levels and localization, but not alternative splicing of cassette exons

Disruptions in connections between the nuclear lamina and nuclear matrix occur in myopathic disorders. However, the biological significance of nuclear lamina - nuclear matrix coupling still remains largely undetermined. Previously it has been demonstrated that the nuclear matrix protein, matrin-3, binds to lamin A/C and this interaction is disrupted in laminopathies resulting in enhanced separation between the lamina and matrix. Matrin-3 has recently been identified as a core regulator of alternative splicing, whereas the involvement of lamin A/C in splicing still remains controversial. In this study, we demonstrate that lamin A/C is not only required for maintaining the nuclear organization of matrin-3, but also of other splicing activators and small nuclear ribonucleoproteins (snRNP) components. Interestingly, mis-localization of these splicing components did not appear to significantly disrupt alternative splicing events of cassette exons regulated by matrin-3. Thus, the lamin A/C-matrin3 interaction is unlikely to be involved in controlling alternative splicing but could be important in coordinating other nuclear activities. Interestingly, matrin-3 knock-down results in misshapen nuclei suggesting its interaction with lamin A/C maybe important in maintaining nuclear structural integrity.

cell biology↗

bioRxiv2018

Heteromeric RNP assembly at LINEs controls lineage-specific RNA processing

It is challenging for RNA processing machineries to select exons within long intronic regions. We find that intronic LINE repeat sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs). This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites in LINEs. Both RBPs repress splicing and 3 end processing within and around LINEs, as demonstrated in cultured human cells and mouse brain. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are confined to deep intronic regions. These RBPs insulate both LINEs and surrounding regions from RNA processing. Upon evolutionary divergence, gradual loss of insulation diversifies the roles of LINEs. Older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for assembly of repressive RBPs, and contribute to evolution of new, lineage-specific transcripts in mammals.

genomics↗