An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

Abstract

In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo, inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei. TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs. K inetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., are transmitted by insect vectors and infect 20 million people, mainly in the most impoverished regions of the world. T. brucei is the causative agent of human African trypanosomiasis, which is a health threat to millions in sub-Saharan Africa and is 100% fatal if left untreated (1). Investigations into the novel biology of kinetoplastids may provide new chemotherapeutic avenues. Indeed, these early-branching eukaryotes utilize unusual processes for gene expression regulation and metabolic organization, and a large number of predicted kinetoplastid proteins lack any homology to proteins in their mammalian hosts.Kinetoplastids are named for their distinctive mitochondrial DNA network, known as a kinetoplast, or kDNA, localized within their single mitochondrion. In T. brucei, kDNA is a giant catenated network of circular molecules comprised of a few dozen copies of a maxicircle (ϳ23 to 40 kb) and approximately 10,000 minicircles (ϳ1 kb) representing over 100 different sequence classes (2, 3). Maxicircles encode 18 mRNAs and 2 ribosomal RNAs. The mitochondrial transcriptome is largely shaped by posttranscriptional processes regulating RNA stability and RNA editing (4-7). RNA editing in kinetoplastids is unique to this group and entails the specific addition and deletion of uridine residues to mRNAs to create translatable open reading frames. The sequence informati...

Gene Loss and Error-Prone RNA Editing in the Mitochondrion of Perkinsela , an Endosymbiotic Kinetoplastid

“…Of 14 other putative editing complex members, only 5 are found, whereas TbRGG1, TbRGG2, MRB8170, and MRB4160 ( 58 , 59 ) are missing in Perkinsela (see Table S1 ). However, TbRGG3, which associates with MRB1 as well as other mitochondrial RNA-binding proteins ( 60 ), yields a hit. Hence, the same picture emerges as for the 20S editosome: the functional core of the MRB1 complex is mostly conserved between Perkinsela and its trypanosomatid relatives.…”

Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing

“…The shift of GAP1 to more dense fractions upon MRB8620 silencing may reflect its increased accumulation on mRNA as described above. Because GAP1 associates with additional proteins outside the MRB1 complex (Madina et al 2014;McAdams et al 2015), these larger GAP1-containing complexes may also comprise non-MRB1 ribonucleoprotein complexes. Similar shifts of GAP1 sedimentation to larger glycerol gradient fractions has been reported previously in response to depletion of specific MRB1 proteins (Aphasizheva et al 2014).…”

Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization

“…In vitro RNA crosslinking was performed as previously described ( 48 , 52 ). Briefly, RNA fragments of A6 mRNA (A6U5, described in ( 56 )) and A6 gRNA (gA6[14]NX, described in ( 56 )) were internally labeled with [α- 32 P]-UTP during in vitro transcription using the T7 Megascript Kit (Ambion).…”