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.

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.

No abstract

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.Transcripts produced by RNA polymerase (RNAP) II are subject to multiple RNA processing steps in the cell nucleus, including capping, splicing, and polyadenylation, before being transported to the cytoplasm as translatable mRNA. Studies over the last decade have indicated an unanticipated integration of these processes (for reviews, see Hirose and Manley 2000;Maniatis and Reed 2002;Proudfoot et al. 2002). While transcription as well as each step of RNA processing can be carried out independently by distinct machineries in vitro, a growing body of evidence, from both biochemical and genetic assays, has revealed that the pre-mRNA processing events, together with mRNA surveillance and mRNA export from the nucleus, are tightly coordinated with transcription ( Fig. 1; for excellent reviews, see Zorio and Bentley 2004;Aguilera 2005a;Reed and Cheng 2005). It has been suggested that such an extensive network of coupling ensures the efficiency and accuracy of each step, and indeed, this undoubtedly is an important aspect of coupling. However, a number of recent studies have revealed a second function for this extensive integration: protecting chromosomes from potentially deleterious effects that can arise from interactions between the nascent RNA and template DNA during transcription. In this review, we present an overview of earlier studies as well as recent findings that support the existence of an evolutionarily conserved role for cotranscriptional processes in the maintenance of genome stability. Our focus will be on how different species protect the integrity of their genomes from potentially disastrous effects of nascent transcripts by diverse mechanisms. We also discuss the possible mechanisms by which, if unchecked, nascent transcripts can lead to genome instability. The reader is also referred to related reviews that have appeared recently (e.g., Aguilera 2002Aguilera , 2005bDrolet 2006). The phenomenon of transcriptional R loopsAn R loop is a structure in which an RNA molecule is partially or completely hybridized with one strand o...

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. Most eukaryotic mRNAs are transcribed as precursors containing noncoding intervening sequences (introns), which are removed by splicing to form the mature mRNA. Recent analyses of the human genome have indicated that alternative splicing is a major source for a large proportion of the disparity between the modest number of genes in the human genome and the much higher complexity of the expressed proteome (Schmutz et al. 2004). Intron removal is catalyzed by a mechanism involving RNA cis-elements and their interaction with a complex repertoire of splicing factors, including snRNPs and non-snRNP proteins (Black 2003). The serine/arginine-rich (SR) protein family is one of the best-studied classes of splicing regulators. SR proteins are involved in multiple steps in the splicing reaction, including U1 snRNP binding to the 5Ј splice site, U2 snRNP binding to the branchpoint sequence at the 3Ј splice site, U1-U2 interactions across the intron or exon, and joining of U4/ 5/6 tri-snRNP to the prespliceosome (Manley and Tacke 1996;Sanford et al. 2003). SR proteins are also capable of influencing splice site selection, largely by binding to regulatory elements in pre-mRNA and recruiting the general splicing machinery to the splicing signals. In contrast to their redundant roles in general splicing, individual SR proteins show diverse RNA binding specificities (Tacke and Manley 1999). Consistent with this, many naturally occurring splicing regulatory elements have been shown to bind specific SR proteins (e.g., see Caputi et al. 2004).While the functions of SR proteins in splicing have been well documented in vitro, elucidation of their roles in vivo has been more difficult. Gene targeting experiments showed that SR proteins are essential for cell viability and/or animal development. In Caenorhabditis eleg...