died processes of chromosome replication, mitosis, and cell division, but also changes in other cellular processes. This hypothesis was supported by our discovery that proteins involved in alternative pre-mRNA splicing are downregulated in S phase. The reason for this apparent systemic regulation of pre-mRNA splicing has yet to be elucidated, but could reflect a need to rapidly alter the isoforms of a cohort of proteins from one cell cycle phase to the next. The depth of our proteome coverage likely reflects changes in the most abundant and readily detectable proteins; thus these fluctuations indicate novel biological pathways and processes that are cell cycleregulated even when the rarest proteins were not quantified. Alternative splicing, 
particularly the production of different isoforms of specific mRNAs at different times in the same cell, is determined by cis elements and the relative concentrations of the trans factors, splicing activators and repressors. Thus, relatively small changes in the concentrations of these common splicing regulatory proteins, particularly the hnRNPs and SR proteins, can result in changes in a number of coordinately regulated alternative splicing events. This study extends and complements the cell cycle proteome analysis by Olsen et al.. Our cells were not only very tightly synchronized in early S phase by the double-thymidine and mitotic shakeoff protocol, but importantly, we collected cells as they progressed synchronously through the cell cycle after release from the block. This protocol is distinct from other popular synchronization methods in which cells were harvested while chemically arrested with replication or mitotic inhibitors or were harvested very shortly after release from such inhibitors. Likely due to these differences, a comparison of proteins that change from G1 to S or from S to G2 in our dataset to those reported by Olsen et al. showed little overlap. Nevertheless, the alternative splicing factors we detected were also reported in the Olsen dataset, although the amplitudes of those changes were less than those we measured. These (S)-(-)-Blebbistatin manufacturer differences may be due to technical variations in culture conditions or to differences in the degree of cell cycle synchrony. One area of close agreement between the two studies, however, is the conclusion that only a subset of cell cycle-regulated changes in protein abundance can be accounted for by changes in mRNA abundance. Although many protein changes detected in this study did not match corresponding changes in mRNA levels, we noted a clear difference between the degree of concordance of 9305921 the mRNA changes and protein changes between the two G1-to-S and S-toG2 datasets. Proteins that increased from S to G2 were more likely to be the products of mRNAs that showed similar cell cycledependent changes, though these mRNA changes were only able to predict,10% of these G2-inducible proteins. This relationship is consistent with the finding that 45% of the cell cycle regulated mRNAs peak in G2/M. Strikingly, more than half of the proteins that changed either increased or decreased from G1 to S phase are among those reported to be polyubiquitinated, but this enrichment was much less or non-significant for proteins that changed from S to G2. 8234901 Taken together, our analysis is consistent with the notion that protein changes from S to G2 are somewhat reflective of changes in mRNA levels, but proteins that change from G1 to S are reflective of ubiquitinmediated protein de