Nucleic Acids Research, 2017 1 doi: 10.1093/nar/gkx963 Nol12 is a multifunctional RNA binding protein at the nexus of RNA and DNA metabolism Daniel D. Scott1,2,†, Christian Trahan1,3,†, Pierre J. Zindy1, Lisbeth C. Aguilar1,Marc Y. Delubac1,3, Eric L. Van Nostrand4, Srivathsan Adivarahan3, Karen E. Wei1, Gene W. Yeo4,5, Daniel Zenklusen3 and Marlene Oeffinger1,2,3,* 1Institut de Recherches Cliniques de Montreal,´ 110 Avenue des Pins Ouest, Montreal,´ Quebec´ H2W 1R7, Canada, 2Faculty of Medicine, Division of Experimental Medicine, McGill University, Montreal,´ Quebec´ H3A 1A3, Canada, 3Departement´ de Biochimie, FacultedeM´ edecine,´ UniversitedeMontr´ eal,´ Montreal,´ Quebec´ H3T 1J4, Canada, 4Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA and 5Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Molecular Engineering Laboratory, A*STAR, Singapore Received February 12, 2016; Revised October 01, 2017; Editorial Decision October 03, 2017; Accepted October 09, 2017 ABSTRACT INTRODUCTION To counteract the breakdown of genome integrity, eu- In eukaryotic cells, the DNA damage response (DDR) com- karyotic cells have developed a network of surveil- prises a network of overlapping cellular signaling path- lance pathways to prevent and resolve DNA dam- ways that detect varied insults to DNA and direct their age. Recent data has recognized the importance of timely and accurate resolution (1). To achieve this, the DDR RNA binding proteins (RBPs) in DNA damage re- must coordinate DNA repair itself with various replica- tive processes including DNA replication, cell growth, cell pair (DDR) pathways. Here, we describe Nol12 as a cycle progression and apoptosis/senescence (1–4). Muta- multifunctional RBP with roles in RNA metabolism tions fin DDR components cause genomic instability and and genome maintenance. Nol12 is found in different a broad spectrum of heritable and spontaneous human dis- subcellular compartments––nucleoli, where it asso- eases (5). Implementation of much of the DDR program ciates with ribosomal RNA and is required for effi- is achieved through transcriptional regulation, both by key cient separation of large and small subunit precur- effector transcription factors such as TP53 and through di- sors at site 2; the nucleoplasm, where it co-localizes rect regulation of RNA polymerases I, II and III (2,6,7). with the RNA/DNA helicase Dhx9 and paraspeck- However, the DDR additionally modulates a large array les; as well as GW/P-bodies in the cytoplasm. Loss of RNA binding proteins (RBPs) to control the synthesis, of Nol12 results in the inability of cells to re- maturation and decay of cellular RNAs (8–11). The DDR cover from DNA stress and a rapid p53-independent regulates both constitutive and transcript-specific splicing through targeting of spliceosomal components and of indi- ATR-Chk1-mediated apoptotic response. Nol12 co- vidual RBPs such as hnRNP K, Sam68, EWSR1, DDX54 localizes with DNA repair proteins in vivo including and SRSF10, respectively (7,12,13). RBPs such as HuR, Dhx9, as well as with TOPBP1 at sites of replica- AUF1 and TIAR modulate mRNA stability in response to tion stalls, suggesting a role for Nol12 in the reso- DDR signaling, as do various miRNAs whose maturation lution of DNA stress and maintenance of genome in- is controlled by the DDR via Dicer (1). HuR also promotes tegrity. Identification of a complex Nol12 interactome, translation of the TP53 mRNA (17). Consistent with these which includes NONO, Dhx9, DNA-PK and Stau1, diverse roles, a number of large-scale genetic and proteomic further supports the protein’s diverse functions in studies of proteins involved in the DDR have shown enrich- RNA metabolism and DNA maintenance, establishing ment for RBPs (2). Nol12 as a multifunctional RBP essential for genome More evidence is emerging, however, that RBPs can go integrity. beyond the paradigm of being DDR effectors and can them- selves participate directly in DNA repair and the DDR *To whom correspondence should be addressed. Tel: +1 514 987 5668; Email: [email protected] †These authors contributed equally to this work as first authors. Present address: Karen E. Wei. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. C The Author(s) 2017. 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 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] Downloaded from https://academic.oup.com/nar/article-abstract/doi/10.1093/nar/gkx963/4561647 by University of california san diego user on 14 November 2017 2 Nucleic Acids Research, 2017 (9,10). Key RNA-regulatory structures within the cell, most Spacer 1 (ITS1), which excised during ribosomal RNA mat- notably the nucleolus and paraspeckles, act as platforms uration, has also been suggested in HeLa cells, concurrent for the regulation and/or assembly of DDR complexes with that of Rrp17 (22). and pathways; fundamental reorganization of these or- Here, we show that the human protein Nol12 is an RNA ganelles is a hallmark of the DDR (3). Several RBPs in- binding protein required for both ribosome maturation and cluding RRP6/EXOSC10, Xrn2, DDX1 and DDX19 are genome integrity in higher eukaryotes. Nol12 is part of early required for preventing the formation of, or resolving aber- 90S and pre-60S ribosomal subunits in vivo, where it is re- rant RNA:DNA hybrids (R-loops) within the genome (4,5). quired for efficient separation of the large and small subunit The multifunctional transcription/translation factor YB- precursors via cleavage at site 2. Knockdown of NOL12 1 is able to bind directly to sites of nucleotide damage impairs cellular proliferation by inducing a G1/S cell cy- and to coordinate repair complex assembly and/or me- cle arrest, but does so outside of a nucleolar stress response tabolize the sites directly (28), while the nucleolar, ribo- and in a p53-independent manner. In addition, Nol12 has some biogenesis proteins NPM1/B23 and NCL/C23 act a putative role in the resolution of DNA stress as loss of as histone chaperones across several DNA repair path- the protein leads to a rapid activation of the DNA dam- ways (6). Numerous other RBPs including FUS/TLS, age response kinase ATR and apoptotic response. More- SFPQ/PSF, NONO/p54nrb, RBM14, RBMX, PRP19, over, Nol12 interacts with chromatin-associated factors, in RPS3 and Dicer––many of which are components of the nu- particular, proteins implicated in DNA damage repair such cleolus and/or paraspeckles––are also recruited to sites of, as the paraspeckle component SFPQ and the RNA/DNA and participate in DNA damage/repair, though the precise DEAD-box helicase Dhx9 involved in genome instability mechanisms of their action(s) have not been fully elucidated prevention, and, furthermore, localizes to sites of replica- (7–9). tion stress and DNA insults in vivo, suggesting a role for In addition to DNA damage, disruption of the accurate Nol12 in the maintenance of genome integrity. maturation and assembly of ribosomes in the nucleolus, a process that involves more than 300 proteins (10), is known MATERIALS AND METHODS to induce G1/S cell cycle arrest via a process termed the ‘nucleolar stress response’ in response to diverse cellular in- DNA constructs sults including transcriptional inhibitors, nutritional stress, NOL12 cDNA was amplified by PCR from pRS414– confluency, as well as the depletion or mutation of various 3xHA-Nol12 (42) with oligonucleotides NdeI-Nol12-F and components of the assembling or mature ribosomes (11,12). XhoI-Nol12-R. The PCR product was cloned into NdeI- In this process, perturbation of pre-ribosomal RNA (pre- XhoI sites of pET21a to generate plasmid pET21a-Nol12– rRNA) synthesis, processing and/or assembly with trans- 6xHis, which was used as template to create a siRNA- acting factors or ribosomal proteins can result in major resistant cDNA of Nol12 by introducing silent muta- structural arrangements of the nucleolus and proteasomal tions by site-directed mutagenesis using the QuickChange degradation of most mature ribosomal proteins, with the Multi Site Mutagenesis Kit (Agilent Technologies) and notable exception of RpL5/uL18 and RpL11/uL5 (13,14). oligos siRNA5-mut, siRNA3–8-mut, siRNA6-mut and These two proteins form a tertiary subcomplex with 5S Link+HA. rRNA which complex protects them from degradation; this In order to construct a PrA-NOL12 encoding plasmid complex subsequently accumulates in the non-ribosomal for Flip-in recombination Flp-In T-REx system, the Nol12 nuclear fraction where it interacts with Mdm2 and prevents cDNA was amplified by PCR from the same template its constitutive ubiquitination and consequent degradation as above with oligonucleotides NcoI-Nol12-F and NotI- of p53 (12–16). While several early papers suggested the Nol12-R, cloned pENTR4 to generate pENTR4-Nol12. existence of independent pathways for other RPs or ribo- Protein A was amplified by PCR from pBXA53 ( )and some biogenesis factors to regulate p53 accumulation, sub- cloned into pFRT-TO-DEST-GFP to generate pFRT-TO- sequent work demonstrated that these pathways in fact act DEST-PrA. pENTR4-Nol12 and pFRT-TO-DEST-PrA via RpL5/RpL11/5S and have underlined the central role were recombined with LR clonase to generate pFRT-TO- of this complex in the induction of cell cycle arrest in re- PrA-Nol12 according to the Gateway system instructions sponse to perturbation of ribosomal biogenesis (14,16,17).