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

Abstract

Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.

Trypanosome RNA editing: the complexity of getting U in and taking U out

“…Mass spectrometry studies by these authors indicate that MRB8620 maintains numerous contacts with the large ribosomal subunit; thus, it may play a more substantial role in post‐editing events than other MRB1 core components. However, a more recent study has shown that RNAi‐mediated repression of MRB8620 indeed results in an inhibition of editing due to the comprised integrity of the MRB1 core, albeit to a lesser degree than when the other studied core MRB1 subunits are downregulated . In this background, transcripts requiring RNA editing accumulated on the GAP1/2 heterotetramer, indicating a disruption of RNA trafficking during the RNA editing process when the MRB1 core is disrupted.…”

“…Indeed, in addition to their roles as integral MRB1 core components, GAP1/2 are present in association with the REH2 RNA helicase and, separately, the TbRGG3 RNA binding protein in the absence of some MRB1 core components, suggesting additional roles for GAP1/2 in mt RNA metabolism in addition to their functions in the MRB1 core. Furthermore, GAP1/2 downregulation does not appear to affect the interaction of the other MRB1 core subunits with each other . The incorporation of GAP1/2 into higher molecular weight complexes, like other MRB1 components, requires mt RNA …”

“…As previously stated, the GAP1/2 component of the MRB1 core binds and stabilizes gRNAs. The functions of other MRB1 core components have been examined in T. brucei engineered for inducible RNAi‐silencing of individual proteins . When gRNA abundance is examined in these cells, it is clear that all other MRB1 core proteins are dispensable for gRNA stabilization.…”

“…The MRB1 core components are essential for growth of PF and, when examined, BF T. brucei . Only under culturing conditions that induces the PF to rely entirely on the mitochondrion for its energy metabolism did MRB8620 downregulation affect flagellate fitness, most likely due to its subtle RNAi phenotype . Interestingly, depletion of most of these proteins also results in a slight increase in gRNA abundance, suggesting that gRNAs are consumed in the editing process (Figure (b)) and, thus, build up when editing is inhibited by MRB1 core protein knockdown.…”

High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing

“…Though RECC is necessary for the editing process, it is not sufficient. Numerous recent studies have demonstrated that a large, possibly dynamic and heterogeneous complex termed MRB1 (mitochondrial RNA binding complex 1) or RESC (RNA editing substrate binding complex) is also essential for RNA editing in kinetoplastids (Fisk et al 2008;Hashimi et al 2008Hashimi et al , 2009Hashimi et al , 2013Weng et al 2008;Acestor et al 2009;Ammerman et al 2011Ammerman et al , 2012Ammerman et al , 2013Kafkova et al 2012;Aphasizheva et al 2014;Huang et al 2015;Madina et al 2015;Read et al 2016). Indeed, the MRB1 complex likely acts as the platform for RNA editing, since it contains readily detectable mRNA and gRNA, while purified RECC lacks associated RNA (Weng et al 2008;Aphasizheva et al 2014;Madina et al 2014Madina et al , 2015.…”