The impact of cytoplasmic capping on transcriptome complexity Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniel E. del Valle-Morales, B.S. Graduate Program in Molecular, Cellular, and Developmental Biology The Ohio State University 2020 Dissertation Committee Daniel R. Schoenberg, Advisor Dawn S. Chandler Ralf Bundschuh Guramrit Singh Copyrighted by Daniel E. del Valle-Morales 2020 Abstract The 5’ cap is an essential modification of mRNAs that is needed for the functionality and lifespan of an mRNA. The cap is added almost immediately after the first nucleotide is transcribed, coordinated by RNGTT and RNMT-RAM bound to the C-terminal tail of RNA Pol II. This process of capping was thought to exclusively occur in the nucleus and loss of the cap was irreversible, leading to the rapid degradation of the mRNA. However, not all mRNAs share this fate. Over the last decade, the Schoenberg lab has characterized cytoplasmic capping, a process where the cap can be restored to previously decapped mRNAs. Cytoplasmic capping is catalyzed by a complex that consists of a cytoplasmic pool of both RNGTT and RNMT-RAM bound to the adapter protein NCK1 along with an unknown 5’ monophosphate kinase. mRNAs that undergo cytoplasmic capping can cycle from being in a decapped state to a recapped state as a way to fine tune gene expression, a process called cap homeostasis. The recapping targets were initially identified using a catalytically inactive and cytoplasmically restricted form of RNGTT termed K294A. Overexpression of K294A resulted in an accumulation of uncapped mRNAs in non-translating mRNPs. A major drawback of this approach was its reliance on detecting uncapped mRNAs, a potentially unstable population that can undergo partial degradation. An alternative tool to study recapping was developed using a catalytically inactive and cytoplasmically restricted form of RNMT termed ΔN-RNMT. ΔN- RNMT expression results in a decrease in the steady state levels of recapped mRNAs. A U2OS stable cell line expressing ΔN-RNMT was developed, and changes in the steady state levels of potential recapping genes were measured by RNA-Seq. 5’ terminal oligopyrimidine (TOP) mRNAs were identified as recapping targets, and I showed direct evidence of recapping i occurring at both the canonical site and downstream in the 5’UTR of eIF3D and eIF3K. Expression of ΔN-RNMT also results in a shift in the 3’UTR usage from proximal sites to distal sites. Uncapped mRNAs that accumulated with inhibition of cytoplasmic capping map to downstream CAGE tags (Kiss et al., 2015). If recapping were to occur at these sites, N- terminally truncated proteins could be translated. In collaboration with the Wysocki lab, we examined the relationship of cytoplasmic capping with N-terminally truncated proteins and showed that half of the downstream N-termini identified decrease when cytoplasmic capping is inhibited. These downstream N-terminal peptides correspond to RNA binding proteins, and the mapped downstream N-termini have a translation start site near the vicinity of the peptide. These studies expand the scope of cytoplasmic capping showing both direct evidence of recapping at canonical and downstream sites, and evidence of cytoplasmic capping expanding the proteome through synthesis of truncated proteins. ii Dedication I can write a thesis in itself for all the people I would like to dedicate this to, but I do want dedicate this to my undergraduate mentor Carlos Ruiz. iii Acknowledgements I first would like to give thanks to my collaborators for their contributions to this work particularly to Bernice Agana for her fantastic work. Thank you to the Center for RNA Biology, the Department of Biological Chemistry and Pharmacology, and the OSU Graduate School for supporting my work with funding and awards and for providing excellent facilities to research and to meet new scientists. Thanks to the NIH for providing the supplemental grant which funded the majority of my research. I would also like to acknowledge my committee members, Ralf, Guramrit, and Dawn, for their tough, yet fair critiques of my work. I would like to thank all of the past lab members; Chandrama, my first lab mentor; Dan Kiss for training me as an RNA biologist, Jackson for his friendship, Shan-Qing, and the various undergraduates Gabe, Andrew, and Mikaela. Thanks to Mike Kearse and his lab for that brief moment that we shared a lab bench. And to Wen Tan for his extensive lab discussions. And thank you, Daniel Schoenberg, for taking me in as an undergraduate during my first research internship. That experience cemented my path towards my Ph.D. and it was a blast to work for you. Gracias por tu ayuda! iv Vita 2011 …………………………………………………………….. First Bilingual Preparatory dd dd School, Aguadilla, Puerto Rico 2014 …………………………………………………………….. B.S. Natural Science, d University of Puerto Rico, d d d Aguadilla 2014 to present …………………………………………………. Graduate Research Associate, d d The Ohio State University Publications del Valle-Morales D., Trotman J., Bundschuh R., Schoenberg D.R. (2020) Inhibition of cytoplasmic cap methylation identifies 5’ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5’ends. Nucleic Acids Res., 48(7), 3806–3815 Fields of study Major Field: Molecular, Cellular, and Developmental Biology v Table of contents Abstract………………………………………………………………………………………….. i Dedication………………………………………………………………………………………. iii Acknowledgements …………………………………………………………………………….. iv Vita ………………………………………………………………………………………………v List of Tables …………………………………………………………………………………. viii List of Figures ………………………………………………………………………………….. ix Chapter 1. Introduction: Recapping of Cytoplasmic mRNAs…………………………………... 1 Abstract…………………………………………………………………………………………. 1 Introduction…………………………………………………………………………………….... 1 The 5’ cap and canonical capping……………………………………………………………... 3 Early Evidence of Cytoplasmic Capping ……………………………………………………... 5 The Cytoplasmic Capping Complex ………………………………………………………….. 6 Characteristics of Recapping Targets …………………………………………………………10 Capping downstream of canonical capping sites …………………………………………….. 13 Unanswered Questions ……………………………………………………………………….. 15 Chapter 2. Inhibition of cytoplasmic cap methylation identifies 5’ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5’ends……………………………… 17 Abstract……………………………………………………………………………………….. 17 Introduction …………………………………………………………………………………... 18 Materials and Methods ……………………………………………………………………….. 20 Results ………………………………………………………………………………………... 27 Discussion ……………………………………………………………………………………..45 Acknowledgements……………………………………………………………………………. 48 Chapter 3. mRNA recapping increases proteome complexity by enabling translation downstream of canonical 5’ends……………………………………………………………………………... 50 Abstract……………………………………………………………………………………… 50 Introduction……………………...……………………………………………………………. 51 vi Materials and Methods ……………………………………………………………………… 52 Results ………………………………………………………………………………………. 61 Discussion …………………………………………………………………………………... 71 Chapter 4. Identification of cytoplasmic capping sites in mRNAs……………………………. 75 Abstract……………………………………………………………………………………… 75 Introduction………………………………………………………………………………….. 75 Cap analysis of gene expression…………………………………………………………….. 78 Cap-SMART………………………………………………………………………………… 81 TeloPrime……………………………………………………………………………………. 85 ReCappable Seq……………………………………………………………………………... 90 Chapter 5. Future Work and concluding remarks……………………………………………... 95 References……………………………………………………………………………………... 99 Appendix List of primers…………………………………………………………………........ 105 vii List of Tables Table 1 PANTHER analysis on ΔN-RNMT downregulated genes ………………………….... 31 Table 2 PANTHER analysis on ΔN-RNMT upregulated genes ………………………............. 41 Table 3 List of primers used in chapter 2……………………………………………………....105 Table 4 List of primers used in chapter 3………………………………………………………106 viii List of Figure Figure 1 Structure of the 5’ cap and the enzymatic steps of nuclear capping ………………….. 2 Figure 2 The cytoplasmic capping complex……………………………………………………. 6 Figure 3 Identification of recapping targets through the inhibition of cytoplasmic capping…… 9 Figure 4 Recapping targets accumulate in non-translating RNPs when cytoplasmic capping is inhibited.........................................................................................................................................10 Figure 5 5’end procession of mRNAs as a possible origin of downstream recapping targets.....13 Figure 6 Validation of ΔN-RNMT cell line………………………….………………………… 26 Figure 7 Metagene analysis of QuantSeq data…………………………………………………. 28 Figure 8 Position of sequence tags and quantitative changes of RNAs after expression of ΔN- RNMT……………………………………………………………………………….…………. 29 Figure 9 Differential expression on TOP mRNAs with ΔN-RNMT induction……………….... 32 Figure 10 Western blot analysis of TOP genes………………………………………………..... 33 Figure 11 Cap-end analysis detects a decrease at the canonical and downstream capping sites of TOP mRNAs……………………………………………………………………………………. 35 Figure 12 Induction of ΔN-RNMT does not activate the cellular stress response……………... 40 Figure 13 3’UTR usage analysis reveals a shift from proximal to distal 3’UTR cleavage sites. 42 Figure 14 PABPN1 protein levels increase with ΔN-RNMT induction……………………….. 44 Figure 15 Inhibition of cytoplasmic capping shows a minor impact in the proteome………… 60 Figure 16 Approach for the enrichment of N-termini generated