bioRxiv preprint doi: https://doi.org/10.1101/471813; this version posted November 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Truncated, uncapped mRNA 5’ ends targeted by cytoplasmic recapping cluster at CAGE tags and some transcripts are alternatively spliced Mikaela R. Berger5,6, Rolando Alvarado1, Daniel L. Kiss1,2,3,4# 1Biomarker Research Program, 2Weil Cornell Medical College 3Institute of Academic Medicine, 4Houston Methodist Cancer Center, Houston Methodist Research Institute, 6670 Bertner Ave, Houston, TX 77030. 5Center for RNA Biology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210 USA; 6Department of Biological Chemistry and Pharmacology, The Ohio State University, 1060 Carmack Rd., Columbus, OH 43210; # corresponding author email: [email protected] Abstract Until cytoplasmic recapping was discovered, decapping was thought to irreversibly destine an mRNA to degradation. Contradicting this idea, we readily observe mRNAs targeted by cytoplasmic capping in uncapped, yet stable forms. 5’ RACE shows that nearly all uncapped ends correspond to CAGE tags and that the recapping of ZNF207 mRNA may be restricted to a single splice isoform. A modified RACE approach detected uncapped 5’ RNA ends mapping to 46 mRNAs in dominant negative cytoplasmic capping enzyme expressing and normal cells. 11 of 46 cloned mRNAs also contained splice isoform-limiting sequences. Collectively, these data reinforce earlier work and suggest that alternative splicing may play a role in targeting transcripts for– and/or determining the position of– cytoplasmic capping. Key Words: cytoplasmic recapping, CAGE, cap homeostasis, alternative splicing, stable uncapped 5’ ends Running Title: Alternative splicing of cytoplasmically recapped mRNAs 1 bioRxiv preprint doi: https://doi.org/10.1101/471813; this version posted November 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abbreviations: m7G, N7-methylguanosine cap; RNMT, RNA guanine-7 methyltransferase; NMD, nonsense-mediated decay; CAGE, capped analysis of gene expression; RACE, rapid amplification of cDNA ends; IPTG, Isopropyl β-D-1- thiogalactopyranoside; X-Gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; K294A, dominant negative cytoplasmic capping enzyme Introduction The N7-methylguanosine (m7G) cap is a critical modification added to the 5’ ends of mRNAs during transcription. Nuclear capping occurs in three steps. First, the triphosphatase domain of RNA guanylyltransferase and 5'-phosphatase (RNGTT, capping enzyme hereafter) removes the gamma phosphate from the first transcribed nucleotide [1]. Capping enzyme then transfers an inverted guanosine residue onto the nascent RNA using its guanylyl transferase domain [1]. Finally, the cap is methylated by RNA guanine-7 methyltransferase (RNMT) [2]. The m7G cap is vital for proper mRNA splicing, processing, packaging, export, and translation [3, 4]. The m7G cap is a focal point of different RNA surveillance pathways and helps protect the mRNA from cellular 5’ to 3’ exonucleases [4]. Although evidence for uncapped mRNA species with mono- and di-phosphate 5’ ends dates back to the 1970’s [5, 6], until recently, decapping was generally thought to irreversibly commit an mRNA to degradation [7, 8]. Certain nonsense-mediated decay (NMD) decay intermediates generated from premature termination codon containing β-globin mRNAs were found to violate this rule [9, 10]. These 5’-truncated mRNA decay intermediates were stable, accumulated in a capped form [9], and required a functional NMD pathway to form [11]. Since NMD occurs exclusively in the cytoplasm, these data alluded to a cytoplasmic RNA recapping mechanism [3, 9]. These capped β-globin decay intermediates remained an 2 bioRxiv preprint doi: https://doi.org/10.1101/471813; this version posted November 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. unexplained oddity for many years; however, the advent of reliable transcriptome-wide methods to assay different RNA populations uncovered the existence of different uncapped RNA species in plants [12, 13] and in mammals [14]. Finally, capped analysis of gene expression (CAGE), a technique designed to identify transcription start sites by tagging the position of m7G caps on mRNAs, showed that nearly 25% of mammalian m7G caps were not at known transcription start sites [15]. Rather, they were located within the body of a transcript and the authors hypothesized mRNAs could be recapped after cleavage or truncation [15]. Cytoplasmic capping was first described around this time [16]. That work identified an RNA capping activity in cytoplasmic extracts and observed that blocking that activity by overexpressing a dominant negative cytoplasmic capping enzyme (K294A) inhibited the cell’s ability to recover from stress [16]. The cytoplasmic capping complex forms when Nck1 interacts with a monophosphate kinase and capping enzyme to coordinate the first two steps of cytoplasmic capping (Figure 1A) [17]. The newly added cap is then methylated by RNMT [18]. Caps added in the cytoplasm appear to be indistinguishable from nuclear-added caps. Roughly 2000 mRNAs are targeted by cytoplasmic capping and that these mRNAs are less frequently associated with translating polysomes when K294A is expressed [19]. Recent work has also shown that uncapped cytoplasmic capping targets retain translationally functional poly(A) tails [20] and that 5’ RACE can detect uncapped ends from 5’ truncated mRNAs in the vicinity of CAGE tags in cells expressing K294A [21]. Collectively, these findings implied that novel 5’ ends from truncated mRNAs should be observable among the non-translating mRNAs in cells where cytoplasmic capping was unimpeded in addition to those where cytoplasmic capping had been blocked. In this report, we readily detected uncapped and 5’ truncated mRNAs in cytoplasmic extracts from uninduced cells, where cytoplasmic capping was normal, in addition to 3 bioRxiv preprint doi: https://doi.org/10.1101/471813; this version posted November 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. those expressing K294A. As before, nearly all of the uncapped ends mapped to CAGE tags. While most of the putative recapping sites yielded sequences that could not discriminate between different splice isoforms, surprisingly, one cluster of 5’ ends was specific to a single isoform of ZNF207 mRNA. To better evaluate this intriguing possibility, we used a dual adaptor RACE approach to identify additional uncapped mRNA ends. Upon closer examination, almost one in four of the cloned mRNA ends contained sequences specific to a limited number of splice isoforms. These findings offer the first hints that sequence cues may help determine the identity of– and/or the position of– cytoplasmic mRNA recapping. Materials and Methods Cell culture and preparation of cytoplasmic extracts. K294A cells generated in [16] were cultured in McCoy’s medium (Gibco) supplemented with 10% tetracycline-tested fetal bovine serum. 150 mm tissue culture dishes were seeded with 3 x 106 log-phase cells. 24h later, expression of K294A was induced by adding doxycycline (1 µg/ml). 24h later, cells were rinsed twice with phosphate buffered saline, scraped from plates with cell lifters, and pelleted by centrifugation at 2500 ×g for 5 min. Cytoplasmic extracts were prepared by lysing pellets in 5 volumes of ice cold cytoplasmic lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM KCl, 10 mM MgCl2, 150 mM NaCl, 0.2 % NP-40, 2 mM DTT, 0.5 mM PMSF, 1 mM sodium orthovanadate, supplemented with 5 µl/ml RNAseOUT (Life Technologies), 25 µl/ml protease inhibitor cocktail (Sigma), 10 µl/ml each phosphatase inhibitor cocktails 2 and 3 (Sigma)) followed by incubation on ice for 10m with gentle agitation every 2m. Nuclei were removed by centrifuging at 16100 ×g for 10m at 4°C and supernatants were used for western blotting and cytoplasmic RNA isolation. Western blotting 4 bioRxiv preprint doi: https://doi.org/10.1101/471813; this version posted November 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Western blots were performed as in [20]. Cytoplasmic extracts were heated to 95°C in 1x Laemmli sample buffer for 5 minutes and then separated on 10% Mini-PROTEAN® TGX™ gels (Bio-Rad) and transferred onto Immobilon-FL PVDF membranes (Millipore). Membranes were blocked with 1% milk in Tris-buffered saline containing 0.05% Tween- 20 (TBS-T), and incubated with rabbit anti-MYC or anti-Tubulin polyclonal antibodies (1:2500 dilution) in 0.1% nonfat milk overnight at 4°C. Blots were washed with TBS-T, incubated with LI-COR IR-680 anti-rabbit secondary antibody (1:10000 dilution) for 2 hours, washed with TBS-T and visualized using Odyssey V.3.0.30 software (LI-COR). Oligonucleotides used in this study All oligonucleotides used in this study are shown in Table S1. Isolation and preparation of cytoplasmic RNA RNA was harvested from cytoplasmic extracts using Direct-Zol kits (Zymo) according to the manufacturer’s reaction clean-up protocol