Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract.
Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-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 degrees 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.
Peris
M
M
Department of Biology, University of California, Los Angeles 90095-662, USA.
Simpson
A M
AM
Grunstein
J
J
Liliental
J E
JE
Frech
G C
GC
Simpson
L
L
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D007891:Leishmania / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry*; D015800:Protozoan Proteins / Q000737:chemistry*; D017393:RNA Editing; D011130:RNA Ligase (ATP) / Q000032:analysis; D012316:RNA Nucleotidyltransferases / Q000032:analysis; D017394:RNA, Guide, Kinetoplastida / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D012261:Ribonucleoproteins / Q000737:chemistry*
D000227:Adenine Nucleotides; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012261:Ribonucleoproteins; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D011130:RNA Ligase (ATP); D017394:RNA, Guide, Kinetoplastida