[PMC free article] [PubMed] [Google Scholar]Volders PJ, Helsens K, Wang X, Menten B, Martens L, Gevaert K, Vandesompele J, and Mestdagh P (2013). shown promise for the treatment of ovarian and breast cancers, as well as other malignancy types (Bitler et al., 2017; Weil and Chen, 2011). PARPi are thought to act by blocking the repair of damaged DNA by PARP-1, thus inducing synthetic lethality in cancers that are deficient in homologous recombination (HR)-mediated DNA repair (e.g., those with inactivating mutations in or mutations or HR-mediated DNA repair deficiency (Bitler et al., 2017; Evans and Matulonis, 2017; Ledermann et al., 2014; Lheureux et al., 2017; Mirza et al., 2016; Scott et al., 2015), suggesting that the role of PARP-1 in DNA damage repair may not be the only reason for its therapeutic potential (Frizzell and Kraus, 2009). Although TOFA PARP-1 is usually upregulated in invasive and malignant tissues from breast cancer patients (Domagala et al., 2011), the precise mechanisms by which PARP-1 supports breast cancer cell growth remain largely unknown. Ribosome biogenesis, which begins in the nucleolus, is usually supported by small nucleolar RNAs (snoRNAs) (Tollervey and Kiss, 1997) and gene-regulating proteins, such as DDX21 (Calo et al., 2015) and PARP-1. snoRNAs are conserved small noncoding RNAs that guideline the processing and site-specific post-transcriptional modification of RNAs (Watkins and Bohnsack, 2012). snoRNA-mediated modifications promote rRNA folding and stability in the nucleolus, thereby playing an essential role in ribosome biogenesis (Jady and Kiss, 2001; Williams and Farzaneh, 2012). The DEAD-box RNA helicase DDX21 has been implicated in ribosome biogenesis, largely through regulation of rDNA transcription at rDNA loci, where it interacts with snoRNAs to regulate rRNA processing and modification (Calo et al., 2015). PARP-1 has been implicated in ribosomal biogenesis through its enrichment in the nucleolus and the ADP-ribosylation (ADPRylation) of several nucleolar proteins, which are required for structural integrity of the nucleolus (Boamah et al., 2012). In addition, PARP-1 has been shown to bind noncoding promoter-associated RNAs (pRNAs), which help establish silent rDNA chromatin and repress rRNA transcription TOFA (Guetg et al., 2012). However, the range of RNA types TOFA bound by PARP-1 and the functional effects of CSF2RA PARP-1-RNA interactions are unknown (e.g., activation of PARP-1 catalytic activity). In addition, TOFA (1) the effects of PARP-1- RNA interactions in the nucleolus, (2) the role of site-specific modification of protein substrates by PARP-1 in ribosome biogenesis, and (3) the biological outcomes of PARP-1-dependent ribosome biogenesis in breast cancers and its potential as a target for PARPi, have not been determined. Here, we show that activation of PARP-1 by snoRNAs, rather than damaged DNA, controls ribosomal DNA (rDNA) transcription, ribosome biogenesis, and protein translation to promote the growth of breast cancer cells. RESULTS RIP-seq Reveals Preferential Binding of PARP-1 to snoRNAs PARP-1 binds broadly to RNAs (Melikishvili et al., 2017), but the specific types of RNA bound by PARP-1 and the cellular effects of the binding in breast cancer are unknown. We used PARP-1-directed RNA immunoprecipitation (IP) coupled with deep sequencing (RIP-seq) performed under native conditions (Zhao et al., 2010) to determine the repertoire of PARP-1-binding RNAs in MCF-7 breast malignancy cells. Using RIP-seq in MCF-7 cells with a PARP-1 antiserum in the absence of UV-induced crosslinking (to avoid PARP-1 activation through DNA damage-induced automodification; Physique S1A) (Zhao et al., 2010), we observed an enrichment of nuclear RNAs in the PARP-1 IP relative to the preimmune (PI) IP (Physique 1A). We prepared and sequenced RIP-seq libraries from immunoprecipitated RNAs and RNA-seq libraries using total input RNA to normalize RIP-seq datasets. The sequencing reads exhibited good correlations between replicates (Physique TOFA S1B). While lncRNAs and mRNAs were the most abundant PARP-1-interacting RNA species, snoRNAs were the most highly enriched (Figures 1B, ?,1C,1C, and S1C). Indeed, nearly.