Published On: Wed, Mar 27th, 2019

Alteration of 28S rRNA 2′-O-methylation by etoposide correlates with decreased SMN phosphorylation and reduced Drosha levels [RESEARCH ARTICLE]

INTRODUCTION

The major types of modifications in human rRNA are pseudouridylation and 2′-O ribose methylation. Human rRNA contains around 100 of each of these modifications, which are performed by small nucleolar ribonucleoproteins (snoRNPs) (Darzacq et al., 2002; Khan and Maden, 1978; Maden et al., 1972; Maden and Salim, 1974; Lafontaine, 2015). SnoRNPs contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. There are two kinds of snoRNPs: box H/ACA, which contain dyskerin and are responsible for the pseudouridylation of rRNA, and box C/D, which contain fibrillarin and perform ribosome methylation of rRNA (Kiss, 2004; Baserga et al., 1991; Fatica et al., 2000; Gautier et al., 1997; Schimmang et al., 1989; Szewczak et al., 2002; Tyc and Steitz, 1989; Watkins et al., 1996). Recent work, coupled with advances in the ability to detect pseudouridylation and 2′-O methylation modifications in a high throughput format, has shown that rRNA modifications can vary, generating ribosome heterogeneity (Birkedal et al., 2015; Lafontaine, 2015; Incarnato et al., 2017; Krogh et al., 2016; Sharma et al., 2017). The presence of a heterogenous pool of ribosomes may allow for the selective increase of a given ‘type’ of ribosome, leading to specialized ribosomes that are optimized for the translation of certain mRNAs (Lafontaine, 2015). Specialized ribosomes have recently been implicated as a major contributor to tumorigenesis (Marcel et al., 2015, 2013; Truitt and Ruggero, 2016).

One possible method that could be used to regulate rRNA modifications, and hence impact ribosome heterogeneity, is to control snoRNP activity. We have published that RNA fragments derived from some small Cajal body-specific RNAs (scaRNAs) may form regulatory RNPs (regRNPs) that influence snoRNP activity (Burke et al., 2018; Poole et al., 2017). As their name implies, scaRNAs accumulate in the Cajal body (CB), which is a subnuclear domain that takes part in the biogenesis of several different classes of RNPs, including small nuclear RNPs (snRNPs). Like rRNA, the small nuclear RNA (snRNA) component of spliceosomal snRNPs requires pseudouridylation and 2′-O ribose methylation modifications for full snRNP functionality (Darzacq et al., 2002; Tycowski et al., 1996; Kiss, 2004; Yu et al., 1998). These modifications in snRNA are guided by the scaRNA component of scaRNPs. (Darzacq et al., 2002; Kiss, 2004). Very interestingly, three scaRNAs (scaRNA 2, 9 and 17) generate nucleolus-enriched fragments of unclear function (Tycowski et al., 2004). We hypothesize that these RNA fragments, and other snoRNAs with uncertain roles, form regulatory RNPs that interact with the snoRNA component of snoRNPs and impact their activity. Therefore, by their interaction with snoRNPs, regRNPs modulate rRNA modifications (Burke et al., 2018; Poole et al., 2017).

Our previous work suggests that proteins enriched in the CB, such as coilin (the CB marker protein), SMN and WRAP53, impact scaRNA 2, 9 and 17 processing (Poole et al., 2016, 2017). SMN is the survival of motor neuron protein, which is mutated in most cases of spinal muscular atrophy and plays important roles in snRNP assembly (Coady and Lorson, 2011; Fischer et al., 1997; Meister et al., 2002; Paushkin et al., 2002; Pellizzoni et al., 1999, 2002). WRAP53 is a scaRNP/telomerase biogenesis factor that interacts with the Cajal body localization signal (CAB box) present in H/ACA scaRNAs and telomerase RNA (Richard et al., 2003; Jády et al., 2004; Mahmoudi et al., 2010; Tycowski et al., 2009; Venteicher et al., 2009; Zhu et al., 2004). In addition to these factors, we reported that Drosha may also contribute to the formation of regulatory RNPs (Logan et al., 2018). Drosha is a member of the RNase III family that initiates microRNA processing (Denli et al., 2004; Lee et al., 2003; Zeng et al., 2005). In the nucleus, Drosha enzymatically cleaves primary-miRNA (pri-miRNA) into the pre-miRNA stem/loop structure that is then transported to the cytoplasm for additional processing by Dicer (Bernstein et al., 2001; Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). Reduction of Drosha alters the fragment to full-length ratio of scaRNA 2 and 9, suggesting that scaRNA 2, 9 and 17 may be unorthodox substrates for Drosha (Logan et al., 2018).

Other conditions that may alter scaRNA 2, 9 and 17 processing are various stresses such as that induced by cisplatin or etoposide (Logan et al., 2018). Notably, we observed that the amount of the mgU2-30 fragment derived from ectopically expressed scaRNA9 is significantly reduced in cells treated with etoposide (Logan et al., 2018). In work presented here, we tested if a subset of rRNA modifications are altered by etoposide treatment. We also examined if scaRNA, snoRNA, SMN and Drosha levels were impacted by etoposide. These studies show that etoposide treatment significantly impacts the phosphorylation profile of SMN and reduces SMN interaction with coilin, resulting in gem formation. Etoposide was also shown to increase the 2′-O-methylation of 28S rRNA at sites 2388 and 3923, which was also found upon Drosha reduction. Collectively, our results demonstrate that stress conditions can influence rRNA modifications and suggest that these alterations may be mediated by changes in snoRNP or regulatory RNP levels.

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