Life stage-specific poly(A) site selection regulated by Trypanosoma brucei DRBD18.

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.

Developmental dynamics of mitochondrial mRNA abundance and editing reveal roles for temperature and the differentiation-repressive kinase RDK1 in cytochrome oxidase subunit II mRNA editing.

IMPORTANCE: Trypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock. The parasite has a complex life cycle consisting of several developmental forms in the human and tsetse fly insect vector. Both the mammalian and insect hosts provide different nutritional environments, so T. brucei must adapt its metabolism to promote its survival and to complete its life cycle. As T. brucei is transmitted from the human host to the fly, the parasite must regulate its mitochondrial gene expression through a process called uridine insertion/deletion editing to achieve mRNAs capable of being translated into functional respiratory chain proteins required for energy production in the insect host. Therefore, it is essential to understand the mechanisms by which T. brucei regulates mitochondrial gene expression during transmission from the mammalian host to the insect vector.

KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing.

Mitochondrial U-indel RNA editing in kinetoplastid protozoa is directed by trans-acting gRNAs and mediated by a holoenzyme with associated factors. Here, we examine the function of the holoenzyme-associated KREH1 RNA helicase in U-indel editing. We show that KREH1 knockout (KO) impairs editing of a small subset of mRNAs. Overexpression of helicase-dead mutants results in expanded impairment of editing across multiple transcripts, suggesting the existence of enzymes that can compensate for KREH1 in KO cells. In depth analysis of editing defects using quantitative RT-PCR and high-throughput sequencing reveals compromised editing initiation and progression in both KREH1-KO and mutant-expressing cells. In addition, these cells exhibit a distinct defect in the earliest stages of editing in which the initiator gRNA is bypassed, and a small number of editing events takes place just outside this region. Wild type KREH1 and a helicase-dead KREH1 mutant interact similarly with RNA and holoenzyme, and overexpression of both similarly disorders holoenzyme homeostasis. Thus, our data support a model in which KREH1 RNA helicase activity facilitates remodeling of initiator gRNA-mRNA duplexes to permit accurate utilization of initiating gRNAs on multiple transcripts.

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing.

Uridine insertion/deletion RNA editing is an extensive post-transcriptional modification of mitochondrial mRNAs in kinetoplastid organisms, including Trypanosoma brucei This process is carried out using trans-acting gRNAs and complex protein machinery. The essential RNA editing substrate binding complex (RESC) serves as the scaffold that modulates protein and RNA interactions during editing, and contains the guide RNA binding complex (GRBC), the RNA editing mediator complexes (REMCs), and organizer proteins. Despite the importance of RESC in editing, the functions of each protein comprising this complex are not completely understood. Here, we further define the roles of a REMC protein, RESC13, and a RESC organizer, RESC14, using high-throughput sequencing on two large pan-edited mRNAs, A6 and COIII. When comparing our analyses to that of a previously published small pan-edited mRNA, RPS12, we find that RESC13 has conserved functions across the three transcripts with regard to editing initiation, gRNA utilization, gRNA exchange, and restricting the formation of long misedited junctions that likely arise from its ability to modulate RNA structure. However, RESC13 does have transcript-specific effects on the types of long junctions whose formation it restricts. RESC14 has a conserved effect on gRNA utilization across the three transcripts analyzed, but has transcript-specific effects on editing initiation, gRNA exchange, and junction formation. Our data suggest that transcript-specific effects of both proteins are due to differences in transcript length and sequences as well as transcript-specific protein interactions. These findings highlight the importance of studying multiple transcripts to determine the function of editing factors.

Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10.

Uridine insertion/deletion editing of mitochondrial mRNAs is a characteristic feature of kinetoplastids, including Trypanosoma brucei. Editing is directed by trans-acting gRNAs and catalyzed by related RNA Editing Core Complexes (RECCs). The non-catalytic RNA Editing Substrate Binding Complex (RESC) coordinates interactions between RECC, gRNA and mRNA. RESC is a dynamic complex comprising GRBC (Guide RNA Binding Complex) and heterogeneous REMCs (RNA Editing Mediator Complexes). Here, we show that RESC10 is an essential, low abundance, RNA binding protein that exhibits RNase-sensitive and RNase-insensitive interactions with RESC proteins, albeit its minimal in vivo interaction with RESC13. RESC10 RNAi causes extensive RESC disorganization, including disruption of intra-GRBC protein-protein interactions, as well as mRNA depletion from GRBC and accumulation on REMCs. Analysis of mitochondrial RNAs at single nucleotide resolution reveals transcript-specific effects: RESC10 dramatically impacts editing progression in pan-edited RPS12 mRNA, but is critical for editing initiation in mRNAs with internally initiating gRNAs, pointing to distinct initiation mechanisms for these RNA classes. Correlations between sites at which editing pauses in RESC10 depleted cells and those in knockdowns of previously studied RESC proteins suggest that RESC10 acts upstream of these factors and that RESC is particularly important in promoting transitions between uridine insertion and deletion RECCs.

Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei.

Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3' to 5' progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

MRB10130 is a RESC assembly factor that promotes kinetoplastid RNA editing initiation and progression.

Uridine insertion deletion editing in kinetoplastid protozoa requires a complex machinery, a primary component of which is the RNA editing substrate binding complex (RESC). RESC contains two modules termed GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex), although how interactions between these modules and their mRNA and gRNA binding partners are controlled is not well understood. Here, we demonstrate that the ARM/HEAT repeat containing RESC protein, MRB10130, controls REMC association with mRNA- and gRNA-loaded GRBC. High-throughput sequencing analyses show that MRB10130 functions in both initiation and 3' to 5' progression of editing through gRNA-defined domains. Editing intermediates that accumulate upon MRB10130 depletion significantly intersect those in cells depleted of another RESC organizer, MRB7260, but are distinct from those in cells depleted of specific REMC proteins. We present a model in which MRB10130 coordinates numerous protein-protein and protein-RNA interactions during editing progression.

Intrinsic and regulated properties of minimally edited trypanosome mRNAs.

Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing. We show that CYb and MURF2 mRNAs exhibit barriers to editing progression that differ from those previously identified for pan-edited mRNAs, primarily at initial gRNA usage and gRNA exchange. We demonstrate that mis-edited junctions arise through multiple pathways including mis-alignment of cognate gRNA, incorrect and sometimes promiscuous gRNA utilization and inefficient gRNA anchoring. We then examined the roles of accessory factors RBP16 and MRP1/2 in maintaining edited CYb and MURF2 populations. RBP16 is essential for initiation of CYb and MURF2 editing, as well as MURF2 editing progression. In contrast, MRP1/2 stabilizes both edited mRNA populations, while further promoting progression of MURF2 mRNA editing. We also analyzed the effects of RNA Editing Substrate Binding Complex components, TbRGG2 and GAP1, and show that both proteins modestly impact progression of editing on minimally edited mRNAs, suggesting a novel function for GAP1.

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

Uridine insertion/deletion RNA editing is an essential process in kinetoplastid parasites whereby mitochondrial mRNAs are modified through the specific insertion and deletion of uridines to generate functional open reading frames, many of which encode components of the mitochondrial respiratory chain. The roles of numerous non-enzymatic editing factors have remained opaque given the limitations of conventional methods to interrogate the order and mechanism by which editing progresses and thus roles of individual proteins. Here, we examined whole populations of partially edited sequences using high throughput sequencing and a novel bioinformatic platform, the Trypanosome RNA Editing Alignment Tool (TREAT), to elucidate the roles of three proteins in the RNA Editing Mediator Complex (REMC). We determined that the factors examined function in the progression of editing through a gRNA; however, they have distinct roles and REMC is likely heterogeneous in composition. We provide the first evidence that editing can proceed through numerous paths within a single gRNA and that non-linear modifications are essential, generating commonly observed junction regions. Our data support a model in which RNA editing is executed via multiple paths that necessitate successive re-modification of junction regions facilitated, in part, by the REMC variant containing TbRGG2 and MRB8180.