František Půta

Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways.

Eukaryotic gene expression requires that all the steps of messenger RNA production are regulated in concert to integrate the diverse inputs cells receive. We discuss the functioning of SNW/SKIP, an essential spliceosomal component and transcriptional coregulator, which may provide regulatory coupling of transcription initiation and splicing. SNW/SKIP potentiates the activity of important transcription factors, such as vitamin D receptor, CBF1 (RBP-Jκ), Smad2/3, and MyoD. It synergizes with Ski in overcoming pRb-mediated cell cycle arrest, and it is targeted by the viral transactivators EBNA2 and E7. SNW/SKIP may aid in conformational transition of the gene expression machine through its avidity to nuclear matrix fractions or by recruiting foldases such as the prolyl isomerase PPIL1. The extensive list of SNW/SKIP partners, its unique primary structure, conserved from yeast to humans, and its essential character suggest a distinct function of general importance.

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. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3′ss distance and masked potential 3′ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3′ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3′ss recognition.

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. Using a synthetic lethality screen, we found that prp45(1–169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2‐associated spliceosomal complexes purified from prp45(1–169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1–169) cells revealed that branch point mutants accumulated more pre‐mRNA whereas 5′ and 3′ splice site mutants showed elevated levels of lariat‐exon intermediate as compared to wild‐type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119–379) in prp45(1–169) cells restored Prp22 partition in the Cwc2‐pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1–169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step‐proofreading helicase Prp22, to splicing efficiency of substrates non‐conforming to the consensus. J. Cell. Biochem. 106: 139–151, 2009.

The homolog of chromatin binding protein Bx42 identified in Dictyostelium.

We identified in Dictyostelium a gene snwA containing a region of similarity to SH2 domains of higher eukaryotes. snwA is homologous to a novel human gene SNW1 and to Bx42 from Drosophila melanogaster, a gene coding for a chromatin binding protein responsive to 20-OH-ecdysone. snwA has one mRNA transcript of an approximate size of 2.5 kb.

Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe

We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

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. We provide evidence supporting their identity as genuine CSL genes. Both cbf11+ and cbf12+ are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTGA/GGAA in vitro. The deletion of cbf11+ is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.

Fungal CSL transcription factors

BackgroundThe CSL (C BF1/RBP-Jκ/S uppressor of Hairless/L AG-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.ResultsWe have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied.ConclusionOur findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.

Molecular characterization of a calmodulin‐like Dictyostelium protein CalB

A gene named calB was cloned and characterized in Dictyostelium. A relationship to calmodulin (CaM) is suggested by sequence identity (50%), similar exon–intron structure and cross‐reactivity with anti‐CaM sera. The level of calB mRNA is developmentally regulated with maxima during aggregation and in spores. CalB null cells grow normally, develop and produce viable spores. We demonstrated the capacity of tagged CalB to bind Ca2+ using the 45Ca2+ overlay assay and showed that its mobility on SDS–PAGE is dependent on Ca2+/EGTA pretreatment.

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. pombe (and Saccharomyces cerevisiae). Three-dimensional, branched, washing-resistant structures composed mostly of elongated, but separate fission yeast cells, were formed within the solid agar medium. The degree of both adhesion and invasion displayed a specific, iron concentration-dependent response. Our results suggest a novel link between iron availability and the intensively studied and important fungal virulence factors, adhesion and invasion.

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. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. Methodology/Principal Findings We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2), we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. Conclusions/Significance This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.