Saccharomyces cerevisiae encodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. Consistent with this observation, we find that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.

During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing.

Splicing is a highly regulated process that depends on numerous factors. It is particularly challenging to quantitatively predict how a mutation will affect precursor messenger RNA (mRNA) structure and the subsequent functional consequences. Here we use a novel Mutational Profiling (-MaP) methodology to obtain highly reproducible endogenous precursor and mature mRNA structural probing data in vivo. We use these data to estimate Boltzmann suboptimal ensembles, and predict the structural consequences of mutations on precursor mRNA structure. Together with a structural analysis of recent cryo-EM spliceosome structures at different stages of the splicing cycle, we determined that the footprint of the Bact complex on precursor mRNA is best able to predict splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene. However, structure alone only achieves 74% accuracy. We therefore developed a β-regression weighting framework that incorporates splice site strength, structure and exonic/intronic splicing regulatory elements which together achieves 90% accuracy for 47 known and six newly discovered splice-altering variants. This combined experimental/computational framework represents a path forward for accurate prediction of splicing related disease-causing variants.

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