The cellular response to highly genotoxic DNA double-strand breaks (DSBs) involves the exquisite coordination of multiple signaling and repair factors. Here, we conducted a functional RNAi screen and identified BAP1 as a deubiquitinase required for efficient assembly of the homologous recombination (HR) factors BRCA1 and RAD51 at ionizing radiation (IR) -induced foci. BAP1 is a chromatin-associated protein frequently inactivated in cancers of various tissues. To further investigate the role of BAP1 in DSB repair, we used a gene targeting approach to knockout (KO) this deubiquitinase in chicken DT40 cells. We show that BAP1-deficient cells are (i) sensitive to IR and other agents that induce DSBs, (ii) defective in HR-mediated immunoglobulin gene conversion, and (iii) exhibit an increased frequency of chromosomal breaks after IR treatment. We also show that BAP1 is recruited to chromatin in the proximity of a single site-specific I-SceI-induced DSB. Finally, we identified six IR-induced phosphorylation sites in BAP1 and showed that mutation of these residues inhibits BAP1 recruitment to DSB sites. We also found that both BAP1 catalytic activity and its phosphorylation are critical for promoting DNA repair and cellular recovery from DNA damage. Our data reveal an important role for BAP1 in DSB repair by HR, thereby providing a possible molecular basis for its tumor suppressor function.

Staufen1 (Stau1) is a ribonucleic acid (RNA)-binding protein involved in the post-transcriptional regulation of gene expression. Recent studies indicate that Stau1-bound messenger RNAs (mRNAs) mainly code for proteins involved in transcription and cell cycle control. Consistently, we report here that Stau1 abundance fluctuates through the cell cycle in HCT116 and U2OS cells: it is high from the S phase to the onset of mitosis and rapidly decreases as cells transit through mitosis. Stau1 down-regulation is mediated by the ubiquitin-proteasome system and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Stau1 interacts with the APC/C co-activators Cdh1 and Cdc20 via its first 88 N-terminal amino acids. The importance of controlling Stau155 levels is underscored by the observation that its overexpression affects mitosis entry and impairs proliferation of transformed cells. Microarray analyses identified 275 Stau155-bound mRNAs in prometaphase cells, an early mitotic step that just precedes Stau1 degradation. Interestingly, several of these mRNAs are more abundant in Stau155-containing complexes in cells arrested in prometaphase than in asynchronous cells. Our results point out for the first time to the possibility that Stau1 participates in a mechanism of post-transcriptional regulation of gene expression that is linked to cell cycle progression in cancer cells.

No abstract

Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.