Inactivation of the SR Protein Splicing Factor ASF/SF2 Results in Genomic Instability

Abstract

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.

Hyperphosphorylation of RNA Polymerase II in Response to Topoisomerase I Cleavage Complexes and Its Association with Transcription- and BRCA1-dependent Degradation of Topoisomerase I

“…44 However, CPT seems to preferentially inhibit Top1-mediated relaxation of positive versus negative DNA supercoiling. 14,45 Finally, CPT may also interfere with RNA splicing by blocking the SR-kinase activity of Top1, 46 which would promote the formation of R-loops 47 and therefore also impair transcriptional elongation. 44,48 Our study (Fig.…”

Regulating the regulators: Serine/arginine‐rich proteins under scrutiny

“…This SR protein protects chromosomal DNA from the detrimental effects of R-loops, which are formed by hybridization between nascent transcripts and their corresponding template DNA during the process of transcription. Thus, SRSF1 depletion leads to DNA doublestrand breaks and genomic instability (33).…”

Graded requirement for the spliceosome in cell cycle progression

“…In contrast, inactivation of the splicing factor ASF/SF2 in chicken cells 29 results in genomic instability, formation of RNA-DNA loops (R-loops), and arrest in the G2 phase of the cell cycle in a splicing-independent manner. Similarly, mouse cells with the spliceosome component PLRG1 deleted show an increase in DNA damage with no evidence of major splicing defects.…”

“…Similarly, mouse cells with the spliceosome component PLRG1 deleted show an increase in DNA damage with no evidence of major splicing defects. 30 However, unlike the G2 arrest observed after ASF/SF2 depletion, 29 PLRG1 deficiency causes an earlier cell cycle arrest, as cells cannot progress through S phase. 30 Hence, a connection between spliceosome depletion and cell cycle defects has been observed after depletion of many different spliceosome subunits.…”

“…17,18 A connection between the spliceosome and cell cycle progression has been found in many organisms including budding yeast, [19][20][21][22][23][24] fission yeast, [25][26][27] Drosophila, 9,28 chicken, 29 mouse, 30 and human cells. 6,11,12,29,31,32 In human cells, depletion of different spliceosome components with siRNAs results in multiple cell cycle defects, with most siRNAs analyzed eliciting mitotic defects 6,11,12,31 although accumulation of cells in S phase 32 has also been observed. The mitotic defects observed after spliceosome depletion have been linked to splicing defects affecting the chromosome cohesion protein sororin 11,12 and Apc2, 11 a subunit of the anaphase-promoting complex/cyclosome (APC/C) required for mitotic progression.…”