TY - JOUR
T1 - Preferential interaction of a 25 kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei
AU - Halbig, Kari
AU - Sacharidou, Anastasia
AU - De Nova-Ocampo, Monica
AU - Cruz-Reyes, Jorge
N1 - Funding Information:
We thank Drs. Linda Guarino and C.T. Ranjith-Kumar for comments on the manuscript and helpful discussions. The antibodies to the MRP complex were kindly provided by Larry Simpson. The viral transcripts H121 and BSK1 were a gift from Cheng C. Kao. Catherine Cifuentes-Rojas kindly assisted with the preparation of the manuscript. This work was supported by a grant from the NIH (GM067130) to J.C.-R.
PY - 2006/10
Y1 - 2006/10
N2 - Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a ∼25 kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [32P] at the first editing site. The ∼25 kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5′-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.
AB - Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a ∼25 kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [32P] at the first editing site. The ∼25 kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5′-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.
KW - Editing complexes
KW - RNA editing
KW - Trypanosoma brucei
KW - mRNA-protein interactions
UR - http://www.scopus.com/inward/record.url?scp=33748445838&partnerID=8YFLogxK
U2 - 10.1016/j.ijpara.2006.05.011
DO - 10.1016/j.ijpara.2006.05.011
M3 - Artículo
SN - 0020-7519
VL - 36
SP - 1295
EP - 1304
JO - International Journal for Parasitology
JF - International Journal for Parasitology
IS - 12
ER -