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.

Berberine is a bioactive isoquinoline alkaloid derived from many plants. Although berberine has been shown to inhibit growth and induce apoptosis of several tumor cell lines, its poor absorption and moderate activity hamper its full therapeutic potential. Here, we describe the synthesis of a series of 9-O-substituted berberine derivatives with improved antiproliferative and apoptosis-inducing activities. An analysis of novel berberine derivatives by EPR spectroscopy confirmed their similar photosensitivity and analogous behavior upon UVA irradiation as berberine, supporting their potential to generate ROS. Improved antitumor activity of novel berberine derivatives was revealed by MTT assay, by flow cytometry and by detection of apoptotic DNA fragmentation and caspase-3 activation, respectively. We showed that novel berberine derivatives are potent inhibitors of growth of HeLa and HL-60 tumor cell lines with IC₅₀ values ranging from 0.7 to 16.7 µM for HL-60 cells and 36 to >200 µM for HeLa cells after 48 h treatment. Further cell cycle analysis showed that the observed inhibition of growth of HL-60 cells treated with berberine derivatives was due to arresting these cells in the G₂/M and S phases. Most strikingly, we found that berberine derivative 3 (9-(3-bromopropoxy)-10-methoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a] isoquinolin-7-ylium bromide) possesses 30-fold superior antiproliferative activity with an IC₅₀ value of 0.7 µM and 6-fold higher apoptosis-inducing activity in HL-60 leukemia cells compared to berberine. Therefore, further studies are merited of the antitumor activity in leukemia cells of this berberine derivative.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

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.

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

Many cellular processes require the activities of complex molecular machines composed of several protein subunits. Insights into these systems can be gained by isolation of protein complexes followed by in vitro analyses determining the identity, posttranslational modifications, and interactions among proteins. Here, we present a protocol for tandem affinity purification (TAP) of protein complexes from the fission yeast Schizosaccharomyces pombe. The protocol employs cells expressing C-terminally TAP-tagged proteins and is suitable for the analysis of purified proteins by mass spectrometry.