Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein

Catherine E. Huang, Sean F. O'Hearn, Barbara Sollner-Webb

Research output: Contribution to journalArticle

Abstract

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Original languageEnglish (US)
Pages (from-to)3194-3203
Number of pages10
JournalMolecular and Cellular Biology
Volume22
Issue number9
DOIs
StatePublished - 2002

Fingerprint

Erythrocyte Anion Exchange Protein 1
RNA Editing
Trypanosoma brucei brucei
Proteins
spleen exonuclease
Trypanosomiasis
Endonucleases
Guide RNA
Double-Stranded RNA
Zinc Fingers
Transferases
RNA Interference
Cell Extracts
Amino Acids
Antibodies
Growth
Genes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein. / Huang, Catherine E.; O'Hearn, Sean F.; Sollner-Webb, Barbara.

In: Molecular and Cellular Biology, Vol. 22, No. 9, 2002, p. 3194-3203.

Research output: Contribution to journalArticle

Huang, Catherine E. ; O'Hearn, Sean F. ; Sollner-Webb, Barbara. / Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein. In: Molecular and Cellular Biology. 2002 ; Vol. 22, No. 9. pp. 3194-3203.
@article{80bc1b50604f439ea77ef362c1c8fbd3,
title = "Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein",
abstract = "Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.",
author = "Huang, {Catherine E.} and O'Hearn, {Sean F.} and Barbara Sollner-Webb",
year = "2002",
doi = "10.1128/MCB.22.9.3194-3203.2002",
language = "English (US)",
volume = "22",
pages = "3194--3203",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "9",

}

TY - JOUR

T1 - Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein

AU - Huang, Catherine E.

AU - O'Hearn, Sean F.

AU - Sollner-Webb, Barbara

PY - 2002

Y1 - 2002

N2 - Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

AB - Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

UR - http://www.scopus.com/inward/record.url?scp=0036235406&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036235406&partnerID=8YFLogxK

U2 - 10.1128/MCB.22.9.3194-3203.2002

DO - 10.1128/MCB.22.9.3194-3203.2002

M3 - Article

C2 - 11940676

AN - SCOPUS:0036235406

VL - 22

SP - 3194

EP - 3203

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 9

ER -