The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7 ؉ (spslu7 ؉ ) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3= splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3= splice site distance, intron length, and the impact of its A/U content at the 5= end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1 ؉ , a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.T he spliceosome, a ribonucleoprotein machinery, comprising five U snRNPs (U1, U2, U4, U5, and U6) and many accessory proteins, performs the precise recognition and removal of introns from primary RNA polymerase II transcripts. The spliceosome undergoes considerable conformational and compositional changes involving protein-protein, RNA-protein, and RNA-RNA interactions to create the catalytic center and carry out the two catalytic reactions. In the first reaction, cleavage at the 5= splice site (5=ss), forms the following intermediates: a lariat intron-3= exon and a 5= exon. In the second reaction, cleavage at the 3=ss, exon ligation and lariat intron excision occur (1). Intronic cis elements (the 5=ss, branch point sequence [BrP], 3=ss, and polypyrimidine tracts [Pyn tracts]) with flanking exonic sequences guide the recognition and alignment of splice sites. These cis elements differ between species and can influence the splicing mechanism (2, 3). Conceivably, concurrent evolution of splicing machineries with genome evolution is evident in divergent groups, such as fungi and metazoans. The relatively short introns, frequent atypically positioned Pyn tracts (between the 5=ss and BrP), and splicing by intron definition are major features that set the fungal splicing machinery apart from that of metazoans (4, 5).Genetic analyses of Saccharomyces cerevisiae and biochemical studies with both yeast and mammalian cell extracts have given functional insights into several spliceosomal factors and snRNPs. In vivo and in vitro studie...

Key Points Sptlc1 is essential for myeloid differentiation during hematopoiesis; ER stress prevents myeloid development in Sptlc1 mutant mice. Accumulation of fatty acid promotes ER stress in Sptlc1 mutant myeloid progenitors.

Edited by Linda SpremulliThe fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Here we perform a domain analysis of the evolutionarily conserved Schizosaccharomyces pombe pre-mRNA-processing factor, SpPrp18. Our mutational and biophysical analyses of the C-terminal ␣-helical bundle reveal critical roles for the conserved region as well as helix five. We generate a novel conditional missense mutant, spprp18-5. To assess the role of SpPrp18, we performed global splicing analyses on cells depleted of prp18 ؉ and the conditional spprp18-5 mutant, which show widespread but intron-specific defects. In the absence of functional SpPrp18, primer extension analyses on a tfIId ؉ intron 1-containing minitranscript show accumulated pre-mRNA, whereas the lariat intron-exon 2 splicing intermediate was undetectable. These phenotypes also occurred in cells lacking both SpPrp18 and SpDbr1 (lariat debranching enzyme), a genetic background suitable for detection of lariat RNAs. These data indicate a major precatalytic splicing arrest that is corroborated by the genetic interaction between spprp18-5 and spprp2-1, a mutant in the early acting U2AF59 protein. Interestingly, SpPrp18 depletion caused cell cycle arrest before S phase. The compromised splicing of transcripts coding for G 1 -S regulators, such as Res2, a transcription factor, and Skp1, a regulated proteolysis factor, are shown. The cumulative effects of SpPrp18-dependent intron splicing partly explain the G 1 arrest upon the loss of SpPrp18. Our study using conditional depletion of spprp18 ؉ and the spprp18-5 mutant uncovers an intron-specific splicing function and early spliceosomal interactions and suggests links with cell cycle progression.Pre-mRNA splicing, a fundamental step in the processing of nascent eukaryotic RNA polymerase II transcripts, achieves precise excision of introns coupled with exon ligation to generate functional mRNAs. The spliceosome, which is composed of five U snRNPs 7 and Ͼ100 auxiliary proteins, assembles onto cis splicing signals, namely the 5Ј splice site (5Јss), the branch point nucleotide, the 3Ј splice site (3Јss), and polypyrimidine (Pyn) tracts. The spliceosomal catalytic core consists of the U2, U5, and U6 snRNPs and accessory proteins. This complex mediates the two trans-esterification reactions required for splicing to occur. First, the 5Јss is cleaved to yield the branched lariat intron-exon 2 and exon 1 intermediates, followed by the second reaction, where the 3Јss is cleaved, the exons are joined, and lariat intron is excised (1, 2).Genetic and biochemical analyses in budding yeast and biochemical studies with mammalian cell extracts have established a network of interactions among factors that act at the second step of splicing (i.e. Prp8, Prp16, Prp17, Prp18, Slu7, and Prp22) (3-8). PRP18, a non-essential budding yeast gene, encodes a U5 snRNP-associated factor. Prp18 has been analyzed extensively in budding yeast and human cell...

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis.

SummaryProperly and effectively managing reference datasets is an important task for many bioinformatics analyses. Refgenie is a reference asset management system that allows to easily organize, retrieve, and share such datasets. Here, we describe the integration of refgenie into the Galaxy platform. Server administrators are able to configure Galaxy to make use of reference datasets made available on a refgenie instance. Additionally, a Galaxy Data Manager tool has been developed to provide a graphical interface to refgenie’s remote reference retrieval functionality. A large collection of reference datasets has also been made available using the CVMFS repository from GalaxyProject.org, with mirrors across the United States, Canada, Europe, and Australia, enabling easy use outside of Galaxy.Availability and implementationThe ability of Galaxy to use refgenie assets was added to the core Galaxy framework in version 20.05, which is available from https://github.com/galaxyproject/galaxy under the Academic Free License version 3.0. The refgenie Data Manager tool can be installed via the Galaxy ToolShed, with source code managed at https://github.com/galaxyproject/tools-iuc/data_managers/data_manager_refgenie_pull and released using an MIT license.