Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 59 and 39 splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.

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BackgroundCbf11 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 FindingsUsing 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. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets.Conclusions/SignificanceOur results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast.

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-Jkappa), 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.

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.