Site-specific proteolytic cleavage prevents ubiquitination and degradation of human REV3L, the catalytic subunit of DNA polymerase ζ.

REV3L, the catalytic subunit of DNA polymerase ζ (Pol ζ), is indispensable for translesion DNA synthesis, which protects cells from deleterious DNA lesions resulting from various intrinsic and environmental sources. However, REV3L lacks a proofreading exonuclease activity and consequently bypasses DNA lesions at the expense of increased mutations, which poses a severe threat to genome stability. Here we report a site-specific proteolytic event of human REV3L. We show that REV3L is cleaved by a threonine aspartase, Taspase1 (TASP1), to generate an N-terminal 70-kDa fragment (N70) and a polypeptide carrying the C-terminal polymerase catalytic domain in human cells. Strikingly, such a post-translational cleavage event plays a vital role in controlling REV3L stability by preventing ubiquitination and proteasome-mediated degradation of REV3L. Indicative of the biological importance of the above REV3L post-translational processing, cellular responses to UV and cisplatin-induced DNA lesions are markedly impaired in human HCT116 cell derivatives bearing defined point mutations in the endogenous REV3L gene that compromise REV3L cleavage. These findings establish a new paradigm in modulating the abundance of REV3L through site-specific proteolysis in human cells.

R-loop-mediated genomic instability is caused by impairment of replication fork progression.

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans.

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1.

Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

A sophisticated mechanism governs Pol ζ activity in response to replication stress.

DNA polymerase ζ (Pol ζ) plays an essential role in replicating damaged DNA templates but contributes to mutagenesis due to its low fidelity. Therefore, ensuring tight control of Pol ζ's activity is critical for continuous and accurate DNA replication, yet the specific mechanisms remain unclear. This study reveals a regulation mechanism of Pol ζ activity in human cells. Under normal conditions, an autoinhibition mechanism keeps the catalytic subunit, REV3L, inactive. Upon encountering replication stress, however, ATR-mediated phosphorylation of REV3L's S279 cluster activates REV3L and triggers its degradation via a caspase-mediated pathway. This regulation confines the activity of Pol ζ, balancing its essential role against its mutations causing potential during replication stress. Overall, our findings elucidate a control scheme that fine tunes the low-fidelity polymerase activity of Pol ζ under challenging replication scenarios.

The RNA binding protein RNPS1 alleviates ASF/SF2 depletion-induced genomic instability.

Formation of transcription-induced R-loops poses a critical threat to genomic integrity throughout evolution. We have recently shown that the SR protein ASF/SF2 prevents R-loop formation in vertebrates by cotranscriptionally binding to nascent mRNA precursors to prevent their reassociation with template DNA. Here, we identify another RNA binding protein, RNPS1, that when overexpressed strongly suppresses the high molecular weight (HMW) DNA fragmentation, hypermutation, and G2 cell cycle arrest phenotypes of ASF/SF2-depleted cells. Furthermore, ablation of RNPS1 by RNA interference in HeLa cells leads to accumulation of HMW DNA fragments. As ASF/SF2 depletion does not influence RNPS1 expression, and RNPS1 cannot compensate for ASF/SF2 function in splicing, our data suggest that RNPS1 is able to function together with ASF/SF2 to form RNP complexes on nascent transcripts, and thereby prevent formation of transcriptional R-loops.

Genotoxic stress causes the accumulation of DNA-dependent protein kinase catalytic subunit phosphorylated at serine 2056 at nuclear speckles and alters pre-mRNA alternative splicing.

RNA splicing has emerged as a critical player in the DNA damage response (DDR). However, the underlying mechanism(s) by which pre-mRNA splicing is coordinately regulated by genotoxic stress has remained largely unclear. Here, we show that a DDR factor, DNA-dependent protein kinase (DNA-PK), participates in the modulation of pre-mRNA splicing in the presence of DNA double-strand break (DSB)-induced genotoxic stress. Through indirect immunostaining, we made the surprising discovery that DNA-PK catalytic subunits (DNA-PKcs) autophosphorylated at serine 2056 (S2056) accumulate at nuclear speckles (dynamic nuclear structures that are enriched with splicing factors), following their dissociation from DSB lesions. Inactivation of DNA-PKcs, either using a small molecule inhibitor or by RNA interference, alters alternative splicing of a set of pre-mRNAs in A549 cells treated with the topoisomerase II inhibitor mitoxantrone, indicative of an involvement of DNA-PKcs in modulating pre-mRNA splicing following genotoxic stress. These findings indicate a novel physical and functional connection between the DNA damage response and pre-mRNA splicing, and enhance our understanding of how mRNA splicing is involved in the cellular response to DSB lesions.

Characterization and proteomic analysis of ovarian cancer-derived exosomes.

Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, because most patients present with advanced disease at diagnosis. Exosomes are important intercellular communication vehicles, released by various cell types. Here we presented firstly the protein profile of highly purified exosomes derived from two ovarian cancer cell lines, OVCAR-3 and IGROV1. The exosomes derived from ovarian cancer cell lines were round and mostly 30-100 nm in diameter when viewed under an electron microscope. The exosomal marker proteins TSG101 and Alix were detected in exosome preparations. The range of density was between 1.09 g/ml and 1.15 g/ml. A total of 2230 proteins were identified from two ovarian cell-derived exosomes. Among them, 1017 proteins were identified in both exosomes including all of the major exosomal protein markers. There were 380 proteins that are not reported in the ExoCarta database. In addition to common proteins from exosomes of various origins, our results showed that ovarian cancer-derived exosomes also carried tissue specific proteins associated with tumorigenesis and metastasis, especially in ovarian carcinoma. Based on the known roles of exosomes in cellular communication, these data indicate that exosomes released by ovarian cancer cells may play important roles in ovarian cancer progression and provide a potential source of blood-based protein biomarkers.

Tumor suppressor RBM5 directly interacts with the DExD/H-box protein DHX15 and stimulates its helicase activity.

RNA binding motif protein 5 (RBM5) is a candidate tumor suppressor gene. Recent studies showed that RBM5 functions as an alternative splicing regulator of apoptosis-related genes. Here, we identify DHX15 and PRP19, two spliceosome components, as novel RBM5-interacting partners. We then show that the G-patch domain of RBM5 is indispensable for its ability to interact with DHX15. Strikingly, we find that RBM5 stimulates the helicase activity of DHX15 in a G patch domain-dependent manner in vitro. Helicase activities play critical roles in modulating pre-mRNA splicing. Our findings thus suggest a new mechanism underlying the regulatory roles of RBM5 in pre-mRNA splicing.

RBM5 promotes exon 4 skipping of AID pre-mRNA by competing with the binding of U2AF65 to the polypyrimidine tract.

Alternative splicing is involved in functional regulation of the mutagenic enzyme activation-induced cytidine deaminase (AID). However, the molecular basis for AID splicing regulation remains undefined. Using a mini-gene-based screen in HeLa cells, we found that overexpression of RNA binding motif protein 5 (RBM5, or LUCA-15/H37) significantly promoted AID exon 4 skipping by suppressing the splicing of intron 3. The inhibitive effect of RBM5 on intron 3 splicing required a weak 3'-splice site (ss). Indicative of the underlying mechanism, RBM5 interfered with the binding of U2AF65 to the polypyrimidine tract at the 3'-ss in vitro. Our findings thus not only shed lights on the regulatory mechanism of AID exon 4 skipping, but also provide new insights into how RBM5 functions in splicing regulation.

Cotranscriptional processes and their influence on genome stability.

Numerous studies support the idea that the complex process of gene expression is composed of multiple highly coordinated and integrated steps. While such an extensive coupling ensures the efficiency and accuracy of each step during the gene expression pathway, recent studies have suggested an evolutionarily conserved function for cotranscriptional processes in the maintenance of genome stability. Specifically, such processes prevent a detrimental effect of nascent transcripts on the integrity of the genome. Here we describe studies indicating that nascent transcripts can rehybridize with template DNA, and that this can lead to DNA strand breaks and rearrangements. We present an overview of the diverse mechanisms that different species employ to keep nascent RNA away from DNA during transcription. We also discuss possible mechanisms by which nascent transcripts impact genome stability, as well as the possibility that transcription-induced genomic instability may contribute to disease.

Alternative splicing and control of apoptotic DNA fragmentation.

It has long been suggested that alternative splicing is involved in regulation of apoptosis by producing mRNA isoforms that encode proteins with distinct and even opposite functions in apoptotic pathways. However, the physiological functions and regulatory mechanisms of such alternative splicing events have been unclear. Recently, it was demonstrated that inactivation of a single SR protein, ASF/SF2, can modulate a specific step in the apoptotic pathway, internucleosomal DNA fragmentation, by regulating ICAD pre-mRNA alternative splicing. These studies have provided new evidence supporting the important role of regulated splicing and SR proteins in the process of apoptosis.

Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.

SR proteins constitute a family of pre-mRNA splicing factors now thought to play several roles in mRNA metabolism in metazoan cells. Here we provide evidence that a prototypical SR protein, ASF/SF2, is unexpectedly required for maintenance of genomic stability. We first show that in vivo depletion of ASF/SF2 results in a hypermutation phenotype likely due to DNA rearrangements, reflected in the rapid appearance of DNA double-strand breaks and high-molecular-weight DNA fragments. Analysis of DNA from ASF/SF2-depleted cells revealed that the nontemplate strand of a transcribed gene was single stranded due to formation of an RNA:DNA hybrid, R loop structure. Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes. Indicative of a direct role, ASF/SF2 prevented R loop formation in a reconstituted in vitro transcription reaction. Our results support a model by which recruitment of ASF/SF2 to nascent transcripts by RNA polymerase II prevents formation of mutagenic R loop structures.

New talents for an old acquaintance: the SR protein splicing factor ASF/SF2 functions in the maintenance of genome stability.

ASF/SF2 is a well-studied SR protein that plays important roles in premRNA splicing and other aspects of RNA metabolism. Genetic inactivation experiments have revealed the fundamental roles of ASF/SF2 and other SR proteins in cell viability and animal development. However, the nature of the events triggered by in vivo depletion of ASF/SF2 remained largely elusive. Recently, we have demonstrated a significant function of ASF/SF2 in the maintenance of genome stability by preventing the formation of R loops, which provided new insights into the essential roles of ASF/SF2 in cellular physiology.

Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation.

ASF/SF2 is an SR protein splicing factor that participates in constitutive and alternative pre-mRNA splicing and is essential for cell viability. Using a genetically modified chicken B-cell line, DT40-ASF, we now show that ASF/SF2 inactivation results in a G2-phase cell cycle arrest and subsequent programmed cell death. However, although several hallmarks of apoptosis are apparent, internucleosomal DNA fragmentation was not detected. Furthermore, inactivation of ASF/SF2 also blocks DNA fragmentation normally induced by a variety of apoptotic stimuli. Notably, mRNA encoding the inhibitor of caspase-activated DNase-L (ICAD-L), which acts as an inhibitor as well as a chaperone of caspase-activated DNase (CAD), decreased in abundance, whereas the level of mRNA encoding ICAD-S, which has only inhibitory activity, increased upon ASF/SF2 depletion. Strikingly, expression of appropriate levels of exogenous human ICAD-L restored apoptotic DNA laddering in ASF/SF2-depleted cells. These results not only indicate that loss of an SR protein splicing factor can induce cell cycle arrest and apoptosis, but also illustrate the important role of ICAD and its regulation by alternative splicing in the process of apoptotic DNA fragmentation.