E-GEOD-47573 - Structural and functional characterization of the N-terminus of Schizosaccharomyces pombe Cwf10

Status
Released on 25 September 2013, last updated on 25 November 2013
Organism
Schizosaccharomyces pombe
Samples (8)
Protocols (3)
Description
The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-∆NTE cells. When the NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10’s overall role facilitating spliceosome rearrangements. Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-ΔNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-ΔNTE
Experiment type
RNA-seq of coding RNA 
Contacts
Melanie Ohi <melanie.ohi@vanderbilt.edu>, Danny A Bitton, Jürg Bähler, Melanie D Ohi, S B Livesay, Scott E Collier
Citation
MINSEQE
Exp. designProtocolsFactorsProcessedSeq. reads
Files
Investigation descriptionE-GEOD-47573.idf.txt
Sample and data relationshipE-GEOD-47573.sdrf.txt
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