the impact of mrna structure on guide rna targeting in kinetoplastid rna editing信使rna结构的影响指导rna针对kinetoplastid rna编辑.pdfVIP

The Impact of mRNA Structure on Guide RNA Targeting in Kinetoplastid RNA Editing 1 2 3 4 1,2,5 Larissa Reifur , Laura E. Yu , Jorge Cruz-Reyes , Michelle vanHartesvelt , Donna J. Koslowsky * 1 Comparative Medicine and Integrative Biology Program, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States of America, 2 Cell and Molecular Biology Program, College of Natural Sciences, Michigan State University, East Lansing, Michigan, United States of America, 3 Department of Biochemistry and Biophysics, Texas AM University, College Station, Texas, United States of America, 4 Dow Corning, Teachers for a New Era, Michigan State University, East Lansing, Michigan, United States of America, 5 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America Abstract Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (59 end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its