Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract

• Published in: Molecular and biochemical parasitology Vol. 85; no. 1; pp. 9 - 24
• Main Authors: Peris, Marian, Simpson, Agda M, Grunstein, Jeremy, Liliental, Joanna E, Frech, Georges C, Simpson, Larry
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.1997
• Subjects: Adenine Nucleotides - metabolism; Animals; ARN; gRNA; Kinetoplastid; LEISHMANIA; Leishmania - chemistry; Leishmania tarentolae; MITOCHONDRIA; Mitochondria - chemistry; MITOCHONDRIE; MITOCONDRIA; NUCLEOPROTEINAS; NUCLEOPROTEINE; NUCLEOPROTEINS; p70; Protozoan Proteins - chemistry; Ribonucleoproteins - chemistry; RNA; RNA Editing; RNA ligase; RNA Ligase (ATP) - analysis; RNA Nucleotidyltransferases - analysis; RNA, Guide - analysis; RNA, Messenger - analysis; RNA, Protozoan - analysis; TUTase; RNA editing; Kinetoplastid; p70; p70, 70 kDa protein; TUTase, terminal uridylyl transferase; nt, nucleotide; TUTase; gRNA; A6, ATPase, subunit 6; TS, cleared supernatant of a Triton X-100 lysed mitochondrial extract; RNA ligase; gRNA, guide RNA; Cyb, cytochrome B; Leishmania; COIII, cytochrome oxidase, subunit III; RPS12, ribosomal protein S12; gCyb-II, gRNA that edits block II of cytochrome b
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(96)02795-8

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [ α- 32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45+p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27°C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45+p50-containing 1800 kDa complex and the p70+gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.