Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in Saccharomyces cerevisiae. However, Prp16 functions are largely unexplored in Schizosaccharomyces pombe, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5ʹ splice site (5'SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5'SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the spprp16F528S mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5'SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in noncoding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in spprp16F528S cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site-snRNA interaction strength.

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and channel substrates to the proteasome. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase resulting in co-degradation of separase along with securin. If true, this establishes Cdc48 as a key regulator of mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

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