3450–3466 Nucleic Acids Research, 2019, Vol. 47, No. 7 Published online 18 January 2019 doi: 10.1093/nar/gkz014 Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins Maddalen Jimenez´ 1,†, Raquel Urtasun1,2,†,Mar´ıa Elizalde1,Mar´ıa Azkona1, M. Ujue Latasa1, Iker Uriarte1,3,Mar´ıa Arechederra1, Diego Alignani2,4,MarinaBarcena-Varela´ 1, Gloria Alvarez-Sola´ 1,3, Leticia Colyn1, Eva Santamar´ıa1,3, Bruno Sangro2,3,5, Carlos Rodriguez-Ortigosa1,2,3, Maite G. Fernandez-Barrena´ 1,2,3, Mat´ıas A. Avila´ 1,2,3,*,‡ and Carmen Berasain 1,2,3,*,‡ 1Hepatology Program, CIMA, University of Navarra, Pamplona 31008, Spain, 2Instituto de Investigaciones Sanitarias de Navarra-IdiSNA, Pamplona 31008, Spain, 3CIBERehd, Instituto de Salud Carlos III, Madrid 28029, Spain, 4Cytometry Unit, CIMA, University of Navarra, Pamplona 31008, Spain and 5Hepatology Unit, Navarra University Clinic, Pamplona 31008, Spain Received July 26, 2018; Revised November 21, 2018; Editorial Decision January 03, 2019; Accepted January 08, 2019 ABSTRACT serve in the cirrhotic liver could be involved in the process of hepatocarcinogenesis. Genome instability is related to disease develop- ment and carcinogenesis. DNA lesions are caused INTRODUCTION by genotoxic compounds but also by the dysregu- lation of fundamental processes like transcription, The preservation of genome integrity is essential for cel- DNA replication and mitosis. Recent evidence indi- lular homeostasis and ultimately for the individual’s sur- cates that impaired expression of RNA-binding pro- vival. DNA lesions, and failure to repair them, can lead teins results in mitotic aberrations and the forma- to a variety of alterations ranging from point mutations to gross chromosomal rearrangements such as deletions, tion of transcription-associated RNA–DNA hybrids translocations, inversions, duplications and even to chro- (R-loops), events strongly associated with DNA in- mosomal numerical changes (aneuploidy) (1). An increased jury. We identify the splicing regulator SLU7 as a key rate of these events is referred to as genome instability, and mediator of genome stability. SLU7 knockdown re- it has been related to the pathogenesis of ageing and neo- sults in R-loops formation, DNA damage, cell-cycle plastic diseases, constituting one of the hallmarks of can- arrest and severe mitotic derangements with loss of cer (2,3). DNA lesions may originate from the action of sister chromatid cohesion (SCC). We define a molec- genotoxic compounds, including xenobiotics, endogenous ular pathway through which SLU7 keeps in check the metabolites and particularly by agents generating reactive generation of truncated forms of the splicing factor oxygen species (4). However, DNA breaks may also occur SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant in association with DNA replication stress caused by a va- negative, or by gain-of-function, SRSF3-TR impair riety of conditions such as the topological characteristics of certain DNA regions, or dysregulated cell proliferation (5). the correct splicing and expression of the splicing / More recently it has also been shown that gene transcription regulator SRSF1 (ASF SF2) and the crucial SCC pro- and alterations in the expression of RNA-binding proteins tein sororin. This unique function of SLU7 was found are important sources of replication stress and genome in- in cancer cells of different tissue origin and also in stability (6–10). the normal mouse liver, demonstrating a conserved We have recently demonstrated that the splicing regulator and fundamental role of SLU7 in the preservation SLU7 is essential to maintain the expression and splicing of of genome integrity. Therefore, the dowregulation of the transcriptome characteristic of the differentiated, qui- SLU7 and the alterations of this pathway that we ob- escent and metabolically functional liver (11). Importantly, we have also found that SLU7 expression is significantly downregulated in damaged liver and in hepatocellular car- *To whom correspondence should be addressed. Tel: +34 948 194700; Fax: +34 948 194717; Email: [email protected] Correspondence may also be addressed to Prof. Mat´ıas A. Avila.´ Tel: +34 948 194700; Fax: +34 948 194717; Email: [email protected] †The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. ‡CB and MAA share senior authorship. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2019, Vol. 47, No. 7 3451 cinoma (HCC) (12), suggesting that SLU7 inhibition may Transfections with siRNAs and microRNAs were per- be causally related to the hepatoinsufficiency observed in formed with Lipofectamine RNAiMAX reagent (Invit- patients with chronic liver injury and in the progression of rogen, CA, USA) following the manufacturer’s instruc- hepatocarcinogenesis (13). Interestingly, we also observed tions. Sequence of siRNAs will be provided upon request. that although reduced, the expression of SLU7 is still re- siSLU7-1 and siSLU7-2 are two independent siRNAs. Indi- tained in human HCC cells and is essential for their sur- vidual siRNAs were used at 75 nM, in co-transfection at 35 vival (14). Mechanistically we found that SLU7 is required nM each and in double transfections siRNAs were used at to maintain the expression of microRNAs generated from 35 nM and microRNAs at 100 nM. pMIR-report plasmids the oncogenic cluster miR-17-92 in order to inhibit apopto- (Ambion, Applied Biosystems, USA) and RNAse H1 plas- sis. Importantly, this function is not restricted to liver cancer mid (RNH1), kindly provided by Dr A. Aguilera (Sevilla, cells, being SLU7 essential for the survival of transformed Spain), were transfected with Lipofectamine 2000 (Invitro- cells of different origin (14). gen, CA, USA) following the manufacturer’s instructions. Intriguingly the dependency on SLU7 for survival found in transformed cells was not observed in normal cells. One Plasmids and luciferase activity of the major differences between normal and transformed cells is their proliferative activity, and the role of spliceo- The sequence corresponding to SRSF3 exon 4 (pEXON4) some components in cell cycle progression has been clearly and the same sequence with three nucleotides mutated established (15–17). In the present work, we demonstrate (QuikChange II Site-Directed Mutagenesis Kit, Stratagene, that SLU7 is essential for cells to progress through mito- CA, USA) at the putative miR-17 seed sequence (pMut), sis and to maintain genome stability. We found that SLU7 were cloned into the pMIR-REPORT luciferase plasmid knockdown results in R-loops accumulation, DNA damage (Ambion, Applied Biosystems). Luciferase activity was de- induction and mitotic errors such as loss of sister chromatid termined using the Dual-Luciferase Reporter Assay System cohesion (SCC). Mechanistically we unravel a molecular (Promega, WI, USA). pathway implicating new truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR), the microRNA miR- Flow cytometry 17 and the regulation of sororin splicing, a central event for normal chromosome segregation (18). Importantly, we Cell-cycle profiles were determined by flow cytometry anal- found that these mechanisms also operate in vivo. In human ysis using a FACS Calibur flow cytometer (BD Bioscience, liver tissue, the downregulation of SLU7 expression is par- CA, USA) and FxCycle™ Violet stain (Thermo Fisher alleled by an increase in the levels of SRSF3-TR isoforms. Scientific, DE, USA). When indicated, cells were treated Altogether, we have gathered in vitro and in vivo evidence overnight with nocodazole (330 nM) (Sigma-Aldrich, MO, showing that SLU7 is essential for the preservation of chro- USA) and collected or washed several times to be collected mosomal stability and DNA integrity during cell prolifera- at different time points after nocodazole release. tion, as well as for mitosis progression. These observations, together with our findings on reduced SLU7 expression in Chromosome spreads liver disease, may contribute to understand the mechanisms of chromosomal instability, which is an early event in hep- Chromosome spreads were prepared as described (21). atocarcinogenesis (19). However, our study also identifies Briefly, cells treated with nocodazole (1 ␮g/ml) for 3 h this splicing regulator as a new molecular target for can- were collected and incubated in 0.075M KCl solution for 20 cer therapy, given its herein demonstrated key function in min, fixed in methanol/acetic acid (3:1 ratio), dropped onto the mitotic progression of cancer cells and the induction of glass slides and stained with 5.8% Giemsa solution (Merck- replication stress and mitotic catastrophe upon its inhibi- Millipore, CA, USA). tion (4,20). Immunofluorescence MATERIALS AND METHODS For immunofluorescence, cells were cultured on cover- Cell culture and transfections slips and 48 h after transfection were fixed with 4% Human HCC cell lines PLC/PRF/5 and HepG2, human paraformaldehyde for 15 min. Immunofluorescence was cervical carcinoma cell line HeLa and human non-small cell performed with anti-alpha-tubulin (Sigma-Aldrich