title: Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA.
abstract: Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: McManus
authors.forename: B K
authors.initials: BK
authors.lastname: Adler
authors.forename: V W
authors.initials: VW
authors.lastname: Pollard
authors.forename: S L
authors.initials: SL
authors.lastname: Hajduk
mesh_terms: D000818:Animals; D001483:Base Sequence; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011072:Poly U / Q000235:genetics*; D017393:RNA Editing; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D016053:RNA, Protozoan / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics*
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012261:Ribonucleoproteins; D011072:Poly U; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D017394:RNA, Guide, Kinetoplastida