CDK1 couples proliferation with protein synthesis.

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Systemic control of protein synthesis through sequestration of translation and ribosome biogenesis factors during severe heat stress.

Environmental stress causes the sequestration of proteins into insoluble deposits including cytoplasmic stress granules (SGs), containing mRNA and a variety of translation factors. Here we systematically identified proteins sequestered in Saccharomyces cerevisiae at 46 °C by a SG co-localization screen and proteomic analysis of insoluble protein fractions. We identified novel SG components including essential aminoacyl-tRNA synthetases. Moreover, we discovered nucleus-associated deposits containing ribosome biogenesis factors. Our study suggests downregulation of cytosolic protein synthesis and nuclear ribosome production at multiple levels through heat shock induced protein sequestrations.

PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.