Abstract
The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1) and the closely related T. brucei RNA Editing Ligase 2 (TbREL2). While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB)-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1) and KREPA1 (for TbREL2). KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441–444) sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.
Highlights
The mitochondrial DNA of trypanosomes contains a structure called the kinetoplast, comprising a large network of concatenated DNA organized into two circular structures [1, 2]
KREPA2 seems to play an important role in regulating the catalytic activities of TbREL1
TbREL1-R372 (38 kDa), similar to the previously reported construct [22], includes the motifs including the five signature motifs (I-V) along with an important arginine 372 that is conserved in TbREL1 and related ligases and is important for catalysis
Summary
The mitochondrial DNA of trypanosomes contains a structure called the kinetoplast, comprising a large network of concatenated DNA organized into two circular structures [1, 2]. Kinetoplast mRNA is first transcribed in an unedited or encrypted form that needs to undergo posttranscriptional processing or “RNA editing” [4] by uridylate (U) insertion or less frequently by uridylate deletion, as specified by the sequence of guide RNAs (gRNAs), to become fully functional RNA. RNA editing is catalyzed by a multiprotein complex called the “editosome,” containing at least 20 different proteins (see reviews [6, 7]). In the first enzymatic step, an editing site-specific endonuclease recognizes the mRNA/gRNA anchor duplex and cleaves the mRNA 30 to the first unpaired nucleotide. The modified mRNA is rejoined by an RNA ligase. This final step is catalyzed by either T. brucei RNA Editing Ligase 1 (TbREL1) or the closely related T. brucei RNA Editing Ligase 2 (TbREL2)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
