Abstract 1430: The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing

Abstract

Abstract (Objective)Recent whole-exome sequencing studies of malignancies have detected recurrent somatic mutations in U2 snRNP components of the spliceosome. These factors have also been listed as novel players of DNA damage response in several genome wide screens and proteome analysis for DDR genes. Although accumulating evidences have implied that the spliceosome plays an important role in genome stability and is an emerging hallmark of carcinogenic pathways, its precise role in genome stability still remains ambiguous. The aim of this study is to clarify the functions of U2 snRNP splicing factors, especially SNRPA1 (Small Nuclear Ribonucleoprotein Polypeptide A1) in DDR pathway. (Methods)SNAPA1 and other U2 snRNP splicing factors were identified as HR repair genes by genome-wide screens based on homologous recombination and RAD51 immunofoci formation. Each splicing factor was depleted by siRNA knockdown in USOS cells. The functions of SNRPA1 in DNA repair were analyzed by HR assay (DR-GFP assay), immunofluorescence, real-time laser micro-irradiation and comet assay (single cell gel electrophoresis). (Results)HR assay showed strong HR deficiencies in splicing factor's depleted cells. In these cells, accumulation of BRCA1 and Rad51, major HR factors, to DNA damage sites were severely impaired. Especially, live cell imaging showed recruitment of SNRPA1 to laser induced DNA damage sites and unveiled its direct involvement to DNA damage repair. Comet assay also showed that depletion of SNRPA1 markedly caused DNA damage with the tail of broken DNA fragments. This DNA damage was R-loop (DNA-RNA hybrid) mediated DNA damage and rescued by overexpression of RNAseH1. (Conclusion) Here we unveiled two distinct pathways how spliceosome U2 snRNP factors contribute to genome stability. The main function is indirect, through transcription, to maintain the protein levels of essential repair factors and contribute to homologous recombination repair. Our data suggest that depletion of splicing factors can be one mechanism for HR deficiency (i.e. BRCAness). In addition real-time laser microirradiation analysis identified the rapid recruitment of SNRPA1 to DNA damage sites. Intensive functional analysis of SNRPA1 unveiled the other, more immediate and direct effect to process R-loop structure, deleterious transcriptional by-products for the genome, at sites of on-going transcription. Citation Format: Michihiro Tanikawa. The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2017-1430