| dc.description.abstract | The spliceosome is a large multi-megadalton RNA-protein machine which facilitates the
removal of introns from eukaryotic nascent pre-mRNAs by two concerted
transesterification reactions. The spliceosome is comprised of five UsnRNPs and several
non snRNP components whose assembly and role in spliceosome catalytic activation have
been well studied in budding yeast S. cerevisiae and mammalian systems. Spliceosome
assembly involves formation of several ordered compositional and structural distinct
complexes a process that is typified by the making and disruption of RNA-RNA, RNAprotein
and protein-protein interactions. This pathway serves to define the splice-sites for
each of the reactions, assist in catalysis and aids in fidelity of splice-site selection. in vitro
studies with budding yeast S. cerevisiae (budding yeast) and mammalian cell free extracts
together with experiments using plasmid expressed mini-transcripts elucidate discrete
functions for DExD/H RNA helicases proteins in spliceosome assembly, catalytic center
formation and disassembly (Staley and Guthrie, 1998). These splicing factors also ensure
the fidelity of splice-site selection by kinetic proofreading of intronic cis elements- the
5’splice-site, the branch consensus sequence and the 3’splice site (Burgess and Guthrie,
1993; Mayas et al., 2006; Xu and Query, 2007; Yang et al., 2013). Thus the DExD/H
proteins are indispensable for the generation of a functional transcriptome.
While the splicing reactions and nearly all factors of the spliceosome are conserved across
eukaryotes, exon-intron architectures vary greatly in diverse species. The short intron
length and degenerate intronic elements are features common to several other fungi and
metazoans. Studies on Schizosacharomyces pombe (fission yeast) which bear such intronic
features and splicing factors with greater sequence similarity to higher eukaryotes than S.
cerevisiae offers the potential to understand a more common splicing mechanism (Kaufer
and Potashkin, 2000; Kuhn and Kaufer, 2003). The spliceosome assembly pathway is
presumably fine-tuned so as recognize and splice introns from pre-mRNAs with very
diverse exon-intron architectures. In S. cerevisiae and humans the splicing factors Slu7 and
Prp18, interact and act at the second splicing reaction for 3’ splice site selection. While in
S. pombe reports from our laboratory have found early splicing roles for SpSlu7 and
SpPrp18, lack of mutual interaction and altered genetic interactions with other
spliceosomal factors (Banerjee et al., 2013; Geetha Melangath, Thesis). Moreover,
mutations of intronic cis elements like the 5’ss dinucleotide GU or in the 3’ss PyAG in at
least a model S. pombe intron arrests splicing prior to first catalysis (Romfo et al, 2000;
Romfo and Wise 1997; Alwarez and Wise 2001). Thus understanding functions for premRNA
splicing of fission yeast will allow for understanding splicing mechanisms relevant
to many fungal introns and metazoans. The DExD/H helicases in the context of fission
yeast splice-site recognition and spliceosomal interactions are largely unexplored. Here we
probed into the role of fission yeast Prp16 by using a combination of molecular, genetic
and biochemical approaches and our key findings are summarized below. We demonstrate
its vital functions in the splicing of a vast majority of S. pombe introns, its interactions with
intronic branch nucleotide and other splicing factors. We also provide compelling
evidences of its influence on other cellular processes like cell cycle progression and
heterochromatinisation. | en_US |
