The CLK1 kinase phosphorylates SR proteins to modulate their splicing regulatory activity. Skipping of alternative exon 4 on the CLK1 pre-mRNA produces a CLK1 variant lacking the catalytic site. Here, we aimed to understand how various SR proteins integrate into the regulatory program that controls CLK1 exon 4 splicing. Previously, we observed that the depletion of SRSF10 promoted the inclusion of CLK1 exon 4. Using expression of tagged proteins and CRISPR/Cas9-mediated knockouts in HCT116 cells, we now identify TRA2b, TRA2a, SRSF4, SRSF5, SRSF7, SRSF8 and SRSF9 as activators of exon 4 inclusion. In contrast, SRSF3, SRSF10 and SRSF12 elicit exon 4 skipping. Using CRISPR/dCas13Rx and RNA immunoprecipitation assays, we map an enhancer in exon 4 interacting with TRA2b. Notably, CLK1 kinase inhibitors antagonized the repressor activity of HA-SRSF10, HA-SRSF12 and HA-SRSF3. Our results suggest that CLK1 exon 4 inclusion is determined primarily by a balance between the activities of TRA2 proteins and CLK-phosphorylated SRSF3. CLK-phosphorylated SRSF10 and SRSF12 would interact with TRA2 proteins to prevent their enhancer activity, allowing SRSF3 to enforce exon 4 skipping more efficiently. Our study provides insight into the complex regulatory network controlling the alternative splicing of CLK1, which uses CLK1-mediated phosphorylation of SR proteins to regulate the inclusion of catalytic exon 4 in CLK1 transcripts.

Temperature-dependent alternative splicing (AS) is a crucial mechanism for organisms to adapt to varying environmental temperatures. In mammals, even slight fluctuations in body temperature are sufficient to drive significant AS changes in a concerted manner. This dynamic regulation allows organisms to finely tune gene expression and protein isoform diversity in response to temperature cues, ensuring proper cellular function and physiological adaptation. Understanding the molecular mechanisms underlying temperature-dependent AS thus provides valuable insights into the intricate interplay between environmental stimuli and gene expression regulation. In this review, we provide an overview of recent advances in understanding temperature-regulated AS across various biological processes and systems. We will discuss the machinery sensing and translating temperature cues into changed AS patterns, the adaptation of the splicing regulatory machinery to extreme temperatures, the role of temperature-dependent AS in shaping the transcriptome, functional implications and the development of potential therapeutics targeting temperature-sensitive AS pathways.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to β-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Alternative splicing of pre-mRNAs is a fundamental step in RNA processing required for gene expression in most metazoans. Serine and arginine-rich proteins (SR proteins) comprise a family of multifunctional proteins that contain an RNA recognition motif (RRM) and the ultra-conserved arginine/serine-rich (RS) domain, and play an important role in precise alternative splicing. Increasing research supports SR proteins as also functioning in other RNA-processing-related mechanisms, such as polyadenylation, degradation, and translation. In addition, SR proteins interact with N6-methyladenosine (m6A) regulators to modulate the methylation of ncRNA and mRNA. Dysregulation of SR proteins causes the disruption of cell differentiation and contributes to cancer progression. Here, we review the distinct biological characteristics of SR proteins and their known functional mechanisms during carcinogenesis. We also summarize the current inhibitors that directly target SR proteins and could ultimately turn SR proteins into actionable therapeutic targets in cancer therapy.

SRSF1 is the founding member of the SR protein family. It is required-interchangeably with other SR proteins-for pre-mRNA splicing in vitro, and it regulates various alternative splicing events. Dysregulation of SRSF1 expression contributes to cancer and other pathologies. Here, we characterized SRSF1\'s interactome using proximity labeling and mass spectrometry. This approach yielded 190 proteins enriched in the SRSF1 samples, independently of the N- or C-terminal location of the biotin-labeling domain. The detected proteins reflect established functions of SRSF1 in pre-mRNA splicing and reveal additional connections to spliceosome proteins, in addition to other recently identified functions. We validated a robust interaction with the spliceosomal RNA helicase DDX23/PRP28 using bimolecular fluorescence complementation and in vitro binding assays. The interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1. During pre-mRNA splicing, DDX23\'s ATPase activity is essential for the pre-B to B spliceosome complex transition and for release of U1 snRNP from the 5\' splice site. We show that the RS-like region of DDX23\'s N-terminal domain is important for spliceosome incorporation, while larger deletions in this domain alter subnuclear localization. We discuss how the identified interaction of DDX23 with SRSF1 and other SR proteins may be involved in the regulation of these processes.

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1\'s kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

Most of mRNAs in Eukaryote were matured after the removal of introns in their pre-mRNA transcripts. Serine/arginine-rich (SR) proteins are a group of splicing regulators regulating the splicing processes globally. Expressions of SR proteins themselves were extensively regulated, at both transcription and splicing levels, under different environmental conditions, specially heat stress conditions. The pine genome is characterized by super-long and easily methylated introns in a large number of genes that derived from the extensive accumulation of transposons (TEs). Here, we identified and analyzed the phylogenetic characteristics of 24 SR proteins and their encoding genes from the pine genome. Then we explored transcription and pre-mRNA splicing expression patterns of SR genes in P. massoniana seedlings under normal and heat stress temperature conditions. Our results showed that the transcription patterns of SR genes in pine exhibited significant changes compared to other plant species, and these changes were not strictly correlated with the intron length and DNA methylation intensity of the SR genes. Interestingly, none of the long introns of SR genes underwent alternative splicing (AS) in our experiment. Furthermore, the intensity of AS regulation may be related to the potential DNA methylation intensity of SR genes. Taken together, this study explores for the first time the characteristics of significant variations in the transcription and splicing patterns of SR proteins in a plant species with an over-accumulation of super-long introns.

Serine/arginine-rich (SR) proteins are key factors with important roles in constitutive and alternative splicing (AS) of pre-mRNAs. However, the role of SR splicing factors in the pathogenicity of T. gondii remains largely unexplored. Here, we investigated the role of splicing factor SR2, a homolog of Plasmodium falciparum SR1, in the pathogenicity of T. gondii. We functionally characterized the predicted SR2 in T. gondii by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The results showed that SR2 was localized in the nucleus and expressed in the tachyzoite and bradyzoite stages. In vitro studies including plaque formation, invasion, intracellular replication, egress and bradyzoite differentiation assays showed that deletion of SR2 in type I RH strain and type II Pru strains had no significant effect on the parasite growth and bradyzoite differentiation (p > 0.05). Interestingly, the disruption of SR2 in RH type I (p < 0.0001) and Pru type II (p < 0.05) strains resulted in varying degrees of attenuated virulence. In addition, disruption of SR2 in type II Pru strain significantly reduced brain cyst burden by ~80% (p < 0.0001). Collectively, these results suggest that splicing factor SR2 is important for the pathogenicity of T. gondii, providing a new target for the control and treatment of toxoplasmosis.

BACKGROUND: Alternative splicing (AS), a posttranscriptional process, contributes to the complexity of transcripts from a limited number of genes in a genome, and AS is considered a great source of genetic and phenotypic diversity in eukaryotes. In animals, AS is tightly regulated during the processes of cell growth and differentiation, and its dysregulation is involved in many diseases, including cancers. Likewise, in plants, AS occurs in all stages of plant growth and development, and it seems to play important roles in the rapid reprogramming of genes in response to environmental stressors. To date, the prevalence and functional roles of AS have been extensively reviewed in animals and plants. However, AS differences between animals and plants, especially their underlying molecular mechanisms and impact factors, are anecdotal and rarely reviewed.
OBJECTIVE: This review aims to broaden our understanding of AS roles in a variety of biological processes and provide insights into the underlying mechanisms and impact factors likely leading to AS differences between animals and plants.
UNASSIGNED: We briefly summarize the roles of AS regulation in physiological and biochemical activities in animals and plants. Then, we underline the differences in the process of AS between plants and animals and especially analyze the potential impact factors, such as gene exon/intron architecture, 5\'/3\' untranslated regions (UTRs), spliceosome components, chromatin dynamics and transcription speeds, splicing factors [serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs)], noncoding RNAs, and environmental stimuli, which might lead to the differences. Moreover, we compare the nonsense-mediated mRNA decay (NMD)-mediated turnover of the transcripts with a premature termination codon (PTC) in animals and plants. Finally, we summarize the current AS knowledge published in animals versus plants and discuss the potential development of disease therapies and superior crops in the future.