Human Nonsense-Mediated RNA Decay Initiates Widely by Endonucleolysis and Targets Snorna Host Genes

Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes Søren Lykke-Andersen,1,4 Yun Chen,2,4 Britt R. Ardal,1 Berit Lilje,2 Johannes Waage,2,3 Albin Sandelin,2 and Torben Heick Jensen1 1Centre for mRNP Biogenesis and Metabolism, Department of Molecular Biology and Genetics, Aarhus University, Aarhus DK-8000, Denmark; 2The Bioinformatics Centre, Department of Biology and Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen DK-2200, Denmark Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we used concurrent transcriptome-wide identification of NMD substrates and their 59–39 decay intermediates to establish that SMG6-catalyzed endonucleolysis widely initiates the degradation of human nonsense RNAs, whereas decapping is used to a lesser extent. We also show that a large proportion of genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, indicating that these RNAs are merely by-products of a primary snoRNA production process. Additionally, transcripts from genes encoding multiple snoRNAs often yield alternative transcript isoforms that allow for differential expression of individual coencoded snoRNAs. Based on our findings, we hypothesize that snoRNA host genes need to be highly transcribed to accommodate high levels of snoRNA production and that the expression of individual snoRNAs and their cognate spliced RNA can be uncoupled via alternative splicing and NMD. [Keywords: RNA decapping; endonucleolytic RNA cleavage; intron-encoded snoRNA; nonsense-mediated decay; regulation of snoRNA expression; snoRNA host genes] Supplemental material is available for this article. Received June 2, 2014; revised version accepted October 3, 2014. All functional transcripts, whether they are protein- 2009; Kelemen et al. 2013). However, many alternative coding (mRNA) or noncoding (ncRNA), are produced as processing options also increase the likelihood of mis- precursor molecules that undergo various processing takes, and, throughout the life span of a transcript, its steps before they take on their final forms. Eukaryotic integrity and functionality is continuously being moni- RNA polymerase II transcribed genes often encode more tored, which ensures that nonfunctional processing by- than one mature RNA species, as exemplified by the products and erroneously processed or outworn mole- alternative splicing of exonic sequences into a variety of cules are degraded by decay machineries residing in either transcript isoforms (Braunschweig et al. 2013; Kornblihtt the nucleus or the cytoplasm (Doma and Parker 2007; et al. 2013), usage of alternative promoters (Carninci et al. Muhlemann and Jensen 2012; Arraiano et al. 2013). For 2006), and the hosting of smaller RNAs, like miRNAs and cytoplasmic RNAs with mRNA-like characteristics, the snoRNAs, within introns (Brown et al. 2008). Regulation distinction between functional and nonfunctional is of such alternative RNA production confers great plas- carried out by means of translation-dependent RNA ticity to eukaryotic gene expression because parameters surveillance systems (Shoemaker and Green 2012; Inada such as expression specificity, stability, localization, and 2013). One such system is the nonsense-mediated RNA protein-coding potential can be altered between tran- decay (NMD) pathway that recognizes and eliminates script isoforms (McGlincy and Smith 2008; Valen et al. RNAs containing premature termination codons (PTCs) Ó 2014 Lykke-Andersen et al. This article is distributed exclusively by 3Present address: Danish Pediatric Asthma Center, Copenhagen Univer- Cold Spring Harbor Laboratory Press for the first six months after the full- sity Hospital, Gentofte DK-2820, Denmark. issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). 4These authors contributed equally to this work. After six months, it is available under a Creative Commons License Corresponding authors: [email protected], [email protected] (Attribution-NonCommercial 4.0 International), as described at http:// Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.246538.114. creativecommons.org/licenses/by-nc/4.0/. 2498 GENES & DEVELOPMENT 28:2498–2517 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/14; www.genesdev.org Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Global mapping of nonsense RNA decay pathways (Kervestin and Jacobson 2012, Schweingruber et al. 2013). Specifically, XRN1 degrades the 39 fragment derived from NMD establishes a quality control system by filtering the endonucleolytically cleaved as well as the decapped away PTC-containing (nonsense) RNAs arising by random full-length nonsense RNA (Gatfield and Izaurralde 2004; errors during early steps of gene expression or genomic Unterholzner and Izaurralde 2004; Huntzinger et al. 2008; mutations and rearrangements. Moreover, NMD plays a Eberle et al. 2009; Arraiano et al. 2013; Nagarajan et al. prominent role in gene expression homeostasis and as part 2013). In Saccharomyces cerevisiae and Drosophila mela- of regulatory responses during, for example, the execution nogaster, the cytoplasmic RNA exosome, a 39–59 exo- of differentiation programs (Kervestin and Jacobson 2012; nuclease, has been implicated in the elimination of fully Schweingruber et al. 2013; Ge and Porse 2014). deadenylated RNA species and the endocleavage-derived Two decades of research has shed light on many aspects 59 fragment, respectively (Mitchell and Tollervey 2003; of NMD, and a current working model can be summa- Gatfield and Izaurralde 2004). rized as follows: Prior to its degradation, a human non- Early reports suggested that human nonsense RNA sense RNA is marked near the PTC by an NMD-eliciting degradation takes place in a way similar to that of the protein complex nucleated around the general NMD yeast S. cerevisiae; i.e., via accelerated decapping and/or factor UPF1 (Kervestin and Jacobson 2012; Schweingruber deadenylation followed by exonucleolysis (Muhlrad and et al. 2013). A stop codon is recognized as being pre- Parker 1994; Cao and Parker 2003; Chen and Shyu 2003; mature when an eRF1/eRF3-bound ribosome stalled at Lejeune et al. 2003; Mitchell and Tollervey 2003; Takahashi the stop codon interacts with UPF1 instead of with the et al. 2003; Couttet and Grange 2004; Yamashita et al. 2005). cytoplasmic polyA tail-binding protein PABPC1, which However, later studies inspired by the observation that normally ‘‘identifies’’ the termination codon as proper nonsense RNAs are exclusively degraded via endocleavage and allows for productive translation termination in D. melanogaster (Gatfield and Izaurralde 2004) revealed (Amrani et al. 2004; Behm-Ansmant et al. 2007; Ivanov that SMG6-catalyzed endocleavage can also occur during et al. 2008; Silva et al. 2008; Singh et al. 2008). UPF1 human NMD (Huntzinger et al. 2008; Eberle et al. 2009). recruitment is favored when the physical distance be- However, the extent to which this contributes to the overall tween the terminating ribosome and the polyA tail is degradation of endogenous nonsense RNAs has been ques- sufficiently long (Behm-Ansmant et al. 2007; Eberle et al. tioned (Yamashita 2013). 2008; Singh et al. 2008) and/or when the stop codon is Here we establish SMG6-catalyzed endocleavage as situated more than ;50 nucleotides (nt) upstream of the a commonly occurring initiating step in human nonsense last splice junction (Nagy and Maquat 1998). The latter RNA decay. Our data suggest that decapping generally mechanism is stimulated as a result of splice junctions serves as a backup option, although it is the preferred being marked by the exon junction complex (EJC), which pathway for a minor subset of substrates. By combining includes the UPF1-interacting proteins UPF3 and UPF2 global identification of nonsense RNAs and their corre- (Kunz et al. 2006; Chamieh et al. 2008; Ivanov et al. 2008; sponding decay intermediates, we identified primary Melero et al. 2012). UPF1 is recruited together with the NMD-responsive isoforms from up to 12% of all expressed kinase complex SMG1C, and subsequent interactions genes. Among these, spliced RNAs derived from both with UPF2 and UPF3 allow SMG1C-mediated phosphor- protein-coding and ‘‘noncoding’’ snoRNA host genes are ylation of UPF1 (Yamashita et al. 2009). This in turn highly enriched. More than 90% of human snoRNA- attracts various factors that can initiate degradation of coding units are situated inside the intronic sequence of the RNA body: (1) The SMG6 endonuclease binds to conventional genes, and the corresponding snoRNA pro- phosphorylated residues in the N-terminal part of UPF1 duction is dependent on the expression of the host gene (Okada-Katsuhata et al. 2012) and catalyzes a cleavage in and the productive splicing of its precursor RNA (Kiss the vicinity of the PTC (Gatfield and Izaurralde 2004; et al. 2006; Brown et al. 2008; Dieci et al. 2009). Our Huntzinger et al. 2008; Eberle et al. 2009); (2) the SMG5/ findings highlight that spliced host gene RNAs are often SMG7 heterodimer binds to phosphorylated

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