Pre-mRNA splicing is a process used by eukaryotic cells to generate messenger RNAs that can be translated into proteins. During splicing, the non-coding regions of the RNAs (introns) are removed from pre-mRNAs and the coding regions (exons) are joined together, resulting in mature mRNAs. The particular steps of splicing are executed by the multimegadalton complex called a spliceosome. This complex is composed of small nuclear ribonucleoproteins, various splicing factors, and other regulatory and auxiliary proteins. In recent years, various post-translational modifications of splicing factors have been shown to contribute significantly to regulation of processes involved in pre-mRNA splicing. In this review, we provide an overview of the most important post-translational modifications of splicing factors that are indispensable for their normal function during pre-mRNA splicing (i.e., phosphorylation, acetylation, methylation, ubiquitination and sumoylation). Moreover, we also discuss how the defects in regulation of splicing factors are related to the development of cancer.
The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.
Protein kinases are important enzymes involved in the regulation of various cellular processes. To function properly, each protein kinase phosphorylates only a limited number of proteins among the thousands present in the cell. This provides a rapid and dynamic regulatory mechanism that controls biological functions of the proteins. Despite the importance of protein kinases, most of their substrates remain unknown. Recently, the advances in the fields of protein engineering, chemical genetics, and mass spectrometry have boosted studies on identification of bona fide substrates of protein kinases. Among the various methods in protein kinase specific substrate identification, genetically engineered protein kinases and quantitative phosphoproteomics have become promising tools. Herein, we review the current advances in the field of chemical genetics in analog-sensitive protein kinase mutants and highlight selected strategies for identifying protein kinase substrates and studying the dynamic nature of protein phosphorylation.
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