title: Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of an mRNA bound to its cognate gRNA.
abstract: Posttranscriptional editing of trypanosome mitochondrial messenger RNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examined. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examined using solution-probing techniques. The mRNA/gRNA crosslinked molecules are efficient substrates for gRNA-directed cleavage. In addition, when the cleavage assay is performed in the presence or absence of additional UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the molecules in a biologically relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide additional support for the predicted secondary structure of interacting gRNA/mRNA pairs.
authors.affiliation: Interdepartmental Graduate Program in Genetics, Michigan State University, East Lansing 48824, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Leung
authors.forename: D J
authors.initials: DJ
authors.lastname: Koslowsky
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000235:genetics*; D001483:Base Sequence; D003573:Cytochrome b Group / Q000235:genetics*; D045303:Cytochromes b; D004722:Endoribonucleases / Q000378:metabolism; D005544:Forecasting; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics*; D017393:RNA Editing; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000077278:RNA, Mitochondrial; D006163:Ribonuclease T1 / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity; D014345:Trypanosoma / Q000235:genetics*
chemical_list: D001059:Apoproteins; D003573:Cytochrome b Group; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D012313:RNA; D045303:Cytochromes b; D004722:Endoribonucleases; C022543:ribonuclease T(2); D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases; D017394:RNA, Guide, Kinetoplastida