Polyadenylation Regulates the Stability of Trypanosoma brucei Mitochondrial RNAs

Abstract

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 pulsechase 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.

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

“…In in vitro decay assays, replacement of four adenosine residues within a 20 nt 3′ tail with a stretch of four uridines did not affect the ability of the 3′ tail to stabilize edited RNA (20). However, the same replacement partially impeded the rapid decay of polyadenylated unedited RNA (19).…”

“…We previously showed that poly(A) tails regulate stability of mitochondrial transcripts in T. brucei (19,20,48). Thus, we wanted to directly determine whether kPAP2 plays a role in regulating RNA abundance in T. brucei mitochondria.…”

“…Moreover, polyadenylation plays a dual role in modulating RNA stability. In in vitro RNA turnover assays, the presence of a poly(A) 20 tail destabilizes unedited RNAs; however, the same modification stabilizes their partially and fully edited counterparts (19,20). In vivo, the 3′ poly(A) tracts on T. brucei mitochondrial RNAs often contain interspersed uridine residues, the distribution of which appears relatively random (18,(21)(22)(23).…”

Human Mitochondrial mRNAs Are Stabilized with Polyadenylation Regulated by Mitochondria-specific Poly(A) Polymerase and Polynucleotide Phosphorylase

“…In trypanosome mitochondria, the length of the poly(A) tail is dependent on the edited status of the mRNA (15)(16)(17). The role of long poly(A) is unknown, whereas short poly(A) seems to stimulate mRNA degradation (18).…”

Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins

“…These include the processing of polycistronic transcripts by 39 and 59 cleavage, transcript maturation through polyadenylation and RNA editing, and RNA turnover (Bhat et al 1992;Militello and Read 1999;Madison-Antenucci et al 2002;Stuart and Panigrahi 2002;Simpson et al 2003;Kao and Read 2005;Lukes et al 2005). These events are controlled by strict regulatory mechanisms for which there are presumably many cis-and trans-acting factors that act to modulate transcript utilization and abundance (Ryan et al 2003;Kao and Read 2005). RNA-binding proteins play a pivotal role in all aspects of cellular RNA processing and utilization (Dreyfuss et al 2002), and several RNA-binding proteins have been described in T. brucei mitochondria, including TBRGG1 (Vanhamme et al 1998), REAP-1 (Madison-Antenucci and Hajduk 2001), RBP16 (Pelletier and Read 2003), and MRP1/2 (Vondruskova et al 2005).…”