Isolation and compositional analysis of trypanosomatid editosomes.
Most mitochondrial (mt) mRNAs in trypanosomes undergo posttranscriptional RNA editing, which inserts and deletes uridines (Us) to produce the mature and functional mRNA. The editing process is catalyzed by multiple enzymatic steps and is carried out by an approximately 20S macromolecular complex, the editosome. Editosomes have been purified from Trypanosoma brucei using various techniques including combinations of column chromatography, gradient sedimentation, monoclonal antibody affinity, and TAP-tag affinity approaches. This article describes in detail the methods for editosome purification and identification of protein components by mass spectrometry analyses. It also describes the methods for isolation and analysis of TAP-tagged mutagenized complexes.
Panigrahi
Aswini K
AK
Seattle Biomedical Research Institute, Seattle, Washington, USA.
Schnaufer
Achim
A
Stuart
Kenneth D
KD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002845:Chromatography / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D046912:Multiprotein Complexes; D021382:Protein Sorting Signals; D021381:Protein Transport; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017393:RNA Editing / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046912:Multiprotein Complexes; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins