Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of ((1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category.

Correction: Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles.

[This corrects the article DOI: 10.1371/journal.pone.

Introns provide a platform for intergenic regulatory feedback of RPL22 paralogs in yeast.

Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs.

Nineteen complex-related factor Prp45 is required for the early stages of cotranscriptional spliceosome assembly.

Splicing in has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169).

Workflow for Genome-Wide Determination of Pre-mRNA Splicing Efficiency from Yeast RNA-seq Data.

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast , about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing.

CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S.

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles.

Background: Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped.

Methodology/principal Findings: Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12.

Fission yeast CSL proteins function as transcription factors.

Background: Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate decisions. CSL proteins recognize specifically the GTG[G/A]AA sequence motif and several mutants compromised in their ability to bind DNA have been reported. In our previous studies we have identified a number of novel putative CSL family members in fungi, organisms lacking the Notch pathway.

Secondary structure is required for 3' splice site recognition in yeast.

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region.

N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast.

Background: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M) can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi.

Stress-induced expression of p53 target genes is insensitive to SNW1/SKIP downregulation.

Pharmacological inhibition of protein kinases that are responsible for the phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II during transcription by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) leads to severe inhibition of mRNA synthesis and activates p53. Transcription of the p53 effectors that are induced under these conditions, such as p21 or PUMA, must bypass the requirement for CTD phosphorylation by the positive elongation factor P-TEFb. Here, we have downregulated SNW1/SKIP, a splicing factor and a transcriptional co-regulator, which was found to interact with P-TEFb and synergistically affect Tat-dependent transcription elongation of HIV 1.

The structure of invadopodia in a complex 3D environment.

Invadopodia and podosomes have been intensively studied because of their involvement in the degradation of extracellular matrix. As both structures have been studied mostly on thin matrices, their commonly reported shapes and characteristics may differ from those in vivo. To assess the morphology of invadopodia in a complex 3D environment, we observed invadopodial formation in cells grown on a dense matrix based on cell-free dermis.

Neoplastic progression of the human breast cancer cell line G3S1 is associated with elevation of cytoskeletal dynamics and upregulation of MT1-MMP.

The newly established breast cancer cell line G3S1, derived from EM-G3 breast cancer progenitors, was analyzed for functional changes related to neoplastic progression manifested by elevated invasiveness and enhanced capability to degrade gelatin. Degradation of gelatin and invasiveness of G3S1 cells was found to be dependent on the activity of matrix proteinases and actin cytoskeletal dynamics. Therefore, the expression and activity of these proteases was compared in G3S1 and EM-G3 cells.

Confocal microscopy reveals Myzitiras and Vthela morphotypes as new signatures of malignancy progression.

G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells.

High environmental iron concentrations stimulate adhesion and invasive growth of Schizosaccharomyces pombe.

We have found that a high iron concentration in solid complete cultivation medium potentiates cell-cell and cell-surface adhesion of the fission yeast Schizosaccharomyces pombe. Spotted giant colonies grown on iron-rich media were found to be more compact and more resistant to washing than those grown on plates with a standard iron content. Furthermore, we have documented that excess environmental iron stimulates the invasive growth of S.

Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division.

The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11+ and cbf12+, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi.

Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates.

Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors.

Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.

Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors.

Fungal CSL transcription factors.

Background: The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins.

Functional mapping of Saccharomyces cerevisiae Prp45 identifies the SNW domain as essential for viability.

The essential gene product Prp45 (379 aa) of Saccharomyces cerevisiae is a highly conserved, but N-terminally abridged, ortholog of the human transcriptional coactivator SKIP, which is involved in TGFbeta, Notch, and steroid hormone signaling. We used a diploid strain harboring PRP45 deletion, which is inviable in the haploid, to test for complementation with the truncated versions of Prp45. The N-terminal half of the protein (aa 1 to 190), denoted as the SNW domain, was found sufficient to support the essential function.

The SH3 domain of Src can downregulate its kinase activity in the absence of the SH2 domain-pY527 interaction.

The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae.