Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1854 PARN – The Tail End Function and mechanisms of specificity and processivity MIKAEL NISSBECK ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0752-7 UPPSALA urn:nbn:se:uu:diva-392998 2019 Dissertation presented at Uppsala University to be publicly examined in B:21, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, Thursday, 31 October 2019 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: professor Renée Schroeder (University of Vienna). Abstract Nissbeck, M. 2019. PARN – The Tail End. Function and mechanisms of specificity and processivity. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1854. 46 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0752-7. Poly(A)-specific ribonuclease (PARN) is an exoribonuclease that is processive, poly(A) specific and cap-binding. PARN deadenylates the poly(A) tails present on a subset of mRNAs and non- coding RNAs, including among others certain snoRNAs, miRNAs and precursor rRNAs. Here, we have investigated molecular mechanisms behind PARN’s specificity for adenine and ribose, essential properties for PARN’s ability to degrade poly(A) on RNAs. We have applied enzyme kinetics and used divalent metal ions as mechanistic probes to show that PARN’s poly(A) specificity is tightly linked to a translocation event during the hydrolytic cycle of PARN action. To further understand the mechanism of PARN’s processive mode of action we are developing a kinetic model that allow us to measure the probability of processive action for each round of the hydrolytic cycle. Our kinetic model will be general and applicable to the processive action of any processive enzymatic activity. In conclusion, our study has so far established a mechanistic link between PARN’s processive mode of action, hydrolytic activity and preference for degrading poly(A). Human patients with genetic lesions in PARN suffer from a spectrum of syndromes called telomere biology disorders (TBD), which are associated with short telomeres. PARN is involved in the maturation of the snoRNA telomerase RNA component (TERC) that is used as template during the elongation of the telomer by the telomerase. Point mutations in the gene for PARN have been identified in patients. We show that point mutations in PARN that perturb its deadenylation activity correlated with TBDs and developmental disorders. Our mechanistic studies of PARN action will provide a framework for our understanding of PARN’s physiological role and in extension the molecular basis for human diseases caused by perturbed PARN action. Our studies will also be of general interest for our detailed and mechanistic understanding of basic and essential mechanisms of gene expression and RNA biology. Keywords: Poly(A) specific ribonuclease, Adenosine, Specificity, Processivty, Telomere biology disorders Mikael Nissbeck, Department of Cell and Molecular Biology, Microbiology, Box 596, Uppsala University, SE-75124 Uppsala, Sweden. © Mikael Nissbeck 2019 ISSN 1651-6214 ISBN 978-91-513-0752-7 urn:nbn:se:uu:diva-392998 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-392998) Mormor List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Nissbeck. M., Henriksson, N., Nilsson. P., Berndtsson, J., Popova, G. and Virtanen, A. (2019) Specificity for adenine is es- tablished during the hydrolytic cycle of poly(A)-specific ribonu- clease (PARN). (Submitted Manuscript) II Nissbeck, M. and Virtanen, A., (2019) Mechanisms of PARN action: Specificity and requirement for the 2’OH groups of the substrate. (Manuscript) III Nissbeck, M., Emmerich, A., Ehrenberg, M. and Virtanen, A. (2019) Mechanisms of PARN action: Kinetic analysis of proces- sive mode of degradation. (Manuscript) IV Dhanraj, A., Gunja, S.M.R., Deveau, A.P., Nissbeck, M., Boonyawat, B., Coombs, A.J., Renieri, A., Mucciolo, A., Ma- rozza, A., Buoni, S., Turner, L., Li, H., Jarrar, A., Sabanayagam, M., Kirby, M., Shago, M., Pinto, D., Berman, J.N., Scherer, S.W., Virtanen, A. and Dror, Y. (2015) Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ri- bonuclease (PARN). Journal of Medical Genetics, 52(11), pp. 738–748. V Dodson, L.M., Baldan, A., Nissbeck, M., Gunja S.M.R., Boonen, P.E., Aubert, G., Birchansky, S., Virtanen, A. and Bertuch. A.A. (2019) From incomplete penetrance with normal telomere length to severe disease and telomere shortening in a family with monoallelic and biallelic PARN pathogenic variants. Human Mutation. John Wiley & Sons, Ltd, (July), p. humu.23898. doi: 10.1002/humu.23898 Reprints were made with permission from the respective publishers. Contents Introduction ...............................................................................................11 Synthesis and maturation of RNAs .........................................................11 mRNA maturation .............................................................................12 Non-coding RNA maturation.............................................................13 Deadenylation .......................................................................................13 mRNA turnover ................................................................................14 PARN – The enzyme .................................................................................16 Structure and domains of PARN ............................................................16 Nuclease domain ...............................................................................17 RNA recognition motif (RRM) ..........................................................18 Specificity .................................................................................................21 Processivity ...............................................................................................25 PARN mutations associated with telomere biology disorders .....................30 Telomeres..............................................................................................30 Telomere biology disorders ...................................................................32 PARN deficiency in patients...................................................................32 Biochemical characterization of the PARN mutation ..............................33 Concluding remarks...................................................................................36 Sammanfattning på Svenska ......................................................................37 Acknowledgements ...................................................................................40 References .................................................................................................41 Abbreviations A Adenosine ATP Adenosine triphosphate C Cytosine Caf1 CCR4-assosiated factor 1 CCR4 carbon catabolite repressor 4 DC Dyskeratosis congenita DcpS Scavenger-decapping enzyme DCP Decapping enzymes DDR DNA damage response EEP exo-endonuclease-phosphatase eIF eukaryotic initiation factor G Guanine HHS Hoyeraal-Hreidersson syndrome IPF idiopathic pulmonary fibrosis mRNA Messenger RNA miRNA microRNA U Uracil PABP Poly(A) binding proteins Pan poly(A) nuclease PAP Poly(A) polymerase PARN poly(A)-specific ribonuclease pre-RNA Precursor RNA RNAP RNA polymerase RRM RNA recognition motif rRNA Ribosomal RNA scaRNA small cajal body specific RNA snRNA spliceosomal RNA snoRNA small nucleolar RNA tRNA Transfer RNA T Thymine TBD Telomer biology disorders TERC Telomeric RNA component TERT Telomeric reverse transcriptase Xrn1 5’-3’ exoribonuclease 1 Introduction The RNA poly(A) tail was discovered by Edmonds, Vaughan and Nakazato (Edmonds, Vaughan and Nakazato, 1971) a decade after the enzymatic func- tion that polymerizes the poly(A) tail on the 3’end of RNA was found (Edmonds and Abrams, 1960). The poly(A) tail is a polymer of adenine nu- cleotides that is polymerized to the 3’end of the RNA by poly(A) polymerase (PAP). The addition of the poly(A) tail is a post-transcriptional modification on RNA. PAP builds the poly(A) tail from adenosine triphosphate (ATP) mol- ecules without a template, unlike transcription or replication of the genome, which both require a template DNA molecule for their synthesis. The polymerization of the poly(A) tail is aided by poly(A) binding proteins (PABP) that also stabilizes the newly formed poly(A) sequences as well as creating a platform for PAP to continue the polymerization of the poly(A) tail (Bienroth, Keller and Wahle, 1993). The polymerization of the mRNA poly(A) tail continues until approximately 200-300 adenines have been incor- porated (Sheets and Wickens, 1989). PABP was first purified and analyzed from polysomes by Blobel (Blobel, 1973). The purified protein associates with the 3’end located poly(A) tail. It has been found that the poly(A)/PABP com- plex makes the polyadenylated RNA resistant to 3’ degradation by RNases. There is a perception of the poly(A) tail that it stabilizes the mRNA. However, the stabilization of the poly(A) tail arises from the binding of PABP to the poly(A) tail and thereby protects the polyadenylated 3’end from nucleases. As a matter of fact, a poly(A) tail in prokaryotes is a signal for degradation of the adenylated RNA, while a free poly(A) tail is readily degraded by deadenylases in the absence of PABP in eukaryotes with one