Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs.
Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K. T., and Read, L. K. (2000) Mol. Cell. Biol. 21, 731-742). In this report, we developed an in vitro assay to directly examine the effects of specific 3'-sequences on RNA degradation. We found that a salt-extracted mitochondrial membrane fraction preferentially degraded polyadenylated mitochondrially and non-mitochondrially encoded RNAs over their non-adenylated counterparts. A poly(A) tail as short as 5 nucleotides was sufficient to stimulate rapid degradation, although an in vivo tail length of 20 adenosines supported the most rapid decay. A poly(U) extension did not promote rapid RNA degradation, and RNA turnover was slowed by the addition of uridine residues to the poly(A) tail. To stimulate degradation, the poly(A) element must be located at the 3' terminus of the RNA. Finally, we demonstrate that degradation of polyadenylated RNAs occurs in the 3' to 5' direction through the action of a hydrolytic exonuclease. These experiments demonstrate that the poly(A) tail can act as a cis-acting element to facilitate degradation of T. brucei mitochondrial mRNAs.
Ryan
Christopher M
CM
Department of Microbiology and the Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214, USA.
Militello
Kevin T
KT
Read
Laurie K
LK
D000241:Adenosine / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D005092:Exonucleases / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011061:Poly A; D011072:Poly U; D026723:Polyadenylation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D012333:RNA, Messenger; D011061:Poly A; D011072:Poly U; D012313:RNA; D005092:Exonucleases; D000241:Adenosine