The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

RESEARCH ARTICLE The RNA encoding the microtubuleassociated protein tau has extensive structure that affects its biology Jonathan L. Chen ID1, Walter N. Moss ID2, Adam Spencer1, Peiyuan Zhang1, Jessica L. Childs-Disney1, Matthew D. Disney ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Chen JL, Moss WN, Spencer A, Zhang P, Childs-Disney JL, Disney MD (2019) The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology. PLoS ONE 14(7): e0219210. https://doi.org/ 10.1371/journal.pone.0219210 Editor: Massimo Caputi, Florida Atlantic University, UNITED STATES Received: April 4, 2019 Accepted: June 18, 2019 Published: July 10, 2019 Copyright: © 2019 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, United States of America, 2 Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa, United States of America * Abstract Tauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimer’s disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within tau’s encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exon-intron junctions and within the 50 and 30 untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 50 UTR contains one structured region, which lies within a known internal ribosome entry site. The 30 UTR contains eight structured regions, including one that contains a polyadenylation signal. A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 30 UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Introduction Funding: This work was supported by the National Institutes of Health (R01-GM097455-07, DP1NS096898, and P01-NS099114 to M.D.D and R00GM112877 to W.N.M) and the Tau Consortium and Rainwater Charitable Foundation (to M.D.D.) as well as startup funds from the Iowa State University College of Agriculture and Life Sciences and the Roy J. Carver Charitable Trust (W.N.M.) RNA structures function in normal cellular processes, such as splicing, protein synthesis, and regulation of gene expression.[1] At the same time, mutations that disrupt RNA structure or formation of ribonucleoproteins (RNPs) can be deleterious and cause disease.[2] This has generated interest in targeting RNA with therapeutics. Evolutionarily conserved RNA structures across species may have common functions.[3] However, the superficial lack of sequence conservation in noncoding RNA (ncRNA) may complicate the search for conserved structures. [4,5] Thus, specialized techniques are needed for discovery of homologous structured regions PLOS ONE | https://doi.org/10.1371/journal.pone.0219210 July 10, 2019 1 / 20 The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology and the Huntington’s Disease Society of America (J.L.C.). Competing interests: The authors have declared that no competing interests exist. Abbreviations: AD, Alzheimer’s disease; APA, alternative polyadenylation; APSI, average pairwise sequence identity; bp, base pair; DMEM, Dulbecco’s Modified Eagle Medium; ED, ensemble diversity; FTDP-17, frontotemporal dementia with parkinsonism-17; htra2β1, transformer 2 beta homolog 1; IRES, internal ribosome entry site; ITAF, IRES trans-acting factor; MAFFT, Multiple Alignment using Fast Fourier Transform; MAPT, microtubule associated protein tau; MBD, microtubule binding domain; MFE, minimum free energy; miRNA, microRNA; mRNA, messenger RNA; MSTD, multiple system tauopathy with presenile dementia; mTOR, mammalian target of rapamycin; ncRNA, noncoding RNA; nt, nucleotide; PD, Parkinson’s disease; RNP, ribonucleoprotein; rRNA, ribosomal RNA; SCI, structure conservation index; SD, standard deviation; SF2, serine and arginine rich splicing factor 1, also known as SRSF1; SHAPE, selective 20 hydroxyl acylation analyzed by primer extension; snRNA, small nuclear RNA; SRp30c, serine and arginine rich splicing factor 9, also known as SRSF9; SRp40, serine and arginine rich splicing factor 5, also known as SRSF5; SRp54, signal recognition particle 54; SRp55, serine and arginine rich splicing factor 6, also known as SRSF6; SVM, support vector machine; UTR, untranslated region. in RNA.[4,6] One method to identify stable, conserved structures combines sequence alignment with thermodynamic-based folding algorithms.[6] Tauopathies are a class of neurodegenerative diseases characterized by the presence of tau inclusion bodies.[7] Tauopathies such as Alzheimer’s and Parkinson’s diseases (AD and PD, respectively) are burdensome socioeconomically and affect more than 35 million and 6.3 million people, respectively, worldwide.[8] Currently available treatments are largely focused on symptoms and do not target underlying disease mechanisms.[7] The tau protein, which binds to microtubules and promotes microtubule assembly and stability, is encoded by the microtubule associated protein tau (MAPT) gene on chromosome 17.[8–11] The MAPT gene is well conserved, with 97 to 100% homology among primates.[12] This 134 kb gene is comprised of 16 exons, among which exons 2, 3, and 10 are known to be alternatively spliced, generating six isoforms ranging from 352 to 441 amino acids in length.[8,10–12] Exons 2 and 3 encode for N-terminal domains while exons 9 to 12 encode microtubule binding domains (MBD).[9] With such complex processing, the MAPT mRNA is likely rich in conserved regulatory structures that may have important functions and may be implicated in tau-associated diseases. Tau proteins bind to and stabilize microtubules via their MBD repeat sequences that interact with negatively charged tubulin residues via their net positive charge.[9] Alterations in the protein coding content of the mRNA, including the number of MBDs, are due to alterna (...truncated)