Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 downregulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.Trypanosoma brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in African wildlife. During their life cycle, T. brucei are transmitted between two different hosts, the tse tse fly insect vector and mammalian host. Due to the resulting drastic changes in environmental growth conditions, the parasite displays differentiationdependent mechanisms of energy metabolism. In the insect midgut stage (procyclic form (PF) 3 ), energy is generated through cytochrome-mediated oxidative phosphorylation, whereas in the mammalian bloodstream form (BF), energy is generated strictly through glycolysis. Correspondingly, the single mitochondrion of T. brucei undergoes extensive alterations in both morphology and gene expression during differentiation. One aspect of this mitochondrial gene regulation is uridine insertion/deletion RNA editing, a process that is restricted to kinetoplastid protozoa, of which T. brucei is a member. During this process, uridine residues are posttranscriptionally added to and/or deleted from pre-mRNAs to produce translatable mature mRNAs. In T. brucei, the accumulation of many edited RNAs is life cycle stage-dependent, with RNAs encoding the cytochrome components apocytochrome b (CYb) and cytochrome oxidase subunit II (COII) edited only in PF and editing of components of the NADH dehydrogenase complex up-regulated i...

Gene expression in the mitochondria of the kinetoplastid parasite Trypanosoma brucei is regulated primarily post-transcriptionally at the stages of RNA processing, editing, and turnover. The mitochondrial RNA-binding complex 1 (MRB1) is a recently identified multiprotein complex containing components with distinct functions during different aspects of RNA metabolism, such as guide RNA (gRNA) and mRNA turnover, precursor transcript processing, and RNA editing. In this study we examined the function of the MRB1 protein, Tb927.5.3010, which we term MRB3010. We show that MRB3010 is essential for growth of both procyclic form and bloodstream form life-cycle stages of T. brucei. Down-regulation of MRB3010 by RNAi leads to a dramatic inhibition of RNA editing, yet its depletion does not impact total gRNA levels. Rather, it appears to affect the editing process at an early stage, as indicated by the accumulation of pre-edited and small partially edited RNAs. MRB3010 is present in large (>20S) complexes and exhibits both RNA-dependent and RNA-independent interactions with other MRB1 complex proteins. Comparison of proteins isolated with MRB3010 tagged at its endogenous locus to those reported from other MRB1 complex purifications strongly suggests the presence of an MRB1 ''core'' complex containing five to six proteins, including MRB3010. Together, these data further our understanding of the function and composition of the imprecisely defined MRB1 complex.

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 59 ends of pan-edited RNAs than at their 39 ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 39 to 59 progression of editing. Detailed analyses of edited RNAs from wildtype and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 39 ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 39 to 59 progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.

A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.Transcription of protein-encoding genes by eukaryotic RNA polymerase II (RNAPII) is a multistep process involving the concerted action of RNAPII and a host of auxiliary proteins (29). One of these auxiliary proteins, the general transcription factor IIF (TFIIF), has been shown to play a functional role during multiple steps of the RNAPII transcription cycle. Biochemical studies have shown that mammalian TFIIF, a heteromeric complex composed of the RAP74 and RAP30 subunits, facilitates the efficient entry of RNAPII into the preinitiation complex (PIC) and is required for the subsequent association of general transcription factors TFIIE and TFIIH (5,7,8,12,27,31). Upon completion of PIC assembly, mammalian TFIIF is reported to induce the wrapping of promoter DNA around RNAPII in the PIC (34) and to facilitate the efficient escape of RNAPII from the promoter subsequent to the formation of the first phosphodiester bond of the nascent transcript (41). Lastly, a number of studies have shown that TFIIF can stimulate the activity of the RNAPII C-terminal domain (CTD) phosphatase Fcp1 (6) and can enhance the efficiency of transcript elongation by interacting with the elongating RNAPII to suppress pausing along the DNA template (1, 2,21,22,26,32,38). Thus, the involvement of TFIIF in many mechanistic steps of the transcription cycle underscores the central importance and complexity of function for this factor.Consistent with the multiple mechanistic roles played by TFIIF, biochemical analyses of higher eukaryotic TFIIF have identified multiple domains in the RAP74 and RAP30 subunits that mediate interactions with ...

Traditional influenza surveillance informs control strategies but can lag behind outbreak onset and undercount cases. Wastewater surveillance is effective for monitoring near real-time dynamics of outbreaks but has not been attempted for influenza. We quantified influenza A virus (IAV) RNA in wastewater during two active outbreaks on university campuses in different parts of the United States and during different times of year using case data from an outbreak investigation and high-quality surveillance data from student athletes. In both cases, the IAV RNA concentrations were strongly associated with reported IAV incidence rates (Kendall's τ values of 0.58 and 0.67 for the University of Michigan and Stanford University, respectively). Furthermore, the RNA concentrations reflected outbreak patterns and magnitudes. For the University of Michigan outbreak, evidence from sequencing IAV RNA from wastewater indicated the same circulating strain identified in cases during the outbreak. The results demonstrate that wastewater surveillance can effectively detect influenza outbreaks and will therefore be a valuable supplement to traditional forms of influenza surveillance.

Our understanding of kinetoplastid RNA (kRNA) editing has centered on this paradigm: guide RNAs (gRNAs) provide a blueprint for uridine insertion/deletion into mitochondrial mRNAs by the RNA editing core complex (RECC). Yet the characterization of constituent subunits of the mitochondrial RNA-binding complex 1 (MRB1) implies that it too is vital to the editing process. The recently elucidated MRB1 architecture will be instrumental in putting functional data from individual subunits into context. Our model depicts two functions for MRB1: mediating multi-round kRNA editing by coordinating the exchange of multiple gRNAs required by RECC to edit lengthy regions of mRNAs, and then linking kRNA editing with other RNA processing events.