title: Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases.
abstract: Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr) Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure.
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Xinlei
authors.initials: X
authors.lastname: Qian
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Fursham M
authors.initials: FM
authors.lastname: Hamid
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Abbas
authors.initials: A
authors.lastname: El Sahili
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Dina Amallia
authors.initials: DA
authors.lastname: Darwis
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Yee Hwa
authors.initials: YH
authors.lastname: Wong
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore.
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore, the Medical Research Council Centre for Developmental Neurobiology, King's College, London SE1 1UL, United Kingdom, and eugene.makeyev@kcl.ac.uk.
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Makeyev
authors.affiliation: From the Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, 138673 Singapore, Singapore, UPMC UMRS CR7-CNRS ERL 8255-INSERM U1135 Centre d' Immunologie et des Maladies Infectieuses, Faculté de Médecine Pierre et Marie Curie, Centre Hospitalier Universitaire Pitié-Salpêtrière, 75031 Paris, France julien@ntu.edu.sg.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
mesh_terms: D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D012324:RNA-Dependent RNA Polymerase
keywords: RNA interference (RNAi); RNA polymerase; RNA-dependent RNA polymerase; electron microscopy (EM); protein evolution; x-ray crystallography