Identification of a spliced leader RNA binding protein from Trypanosoma cruzi.
Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.
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Department of Microbiology and Immunology, Medical College of Virginia Campus, Box 980678, Virginia Commonwealth University, 1101 East Marshall, Rm. 5036 Sanger Hall, Richmond, VA 23298-0678, USA.
Wen
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Benegal
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Wang
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Buck
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D020040:Trans-Splicing; D015533:Transcriptional Activation; D014349:Trypanosoma cruzi / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D015800:Protozoan Proteins; D016053:RNA, Protozoan; D020038:RNA, Spliced Leader; D016601:RNA-Binding Proteins; C435212:XB1 protein, Trypanosoma cruzi