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.

Adaptation to host temperature is a prerequisite for any pathogen capable of causing deep infection in humans. Our previous studies demonstrated that a Cryptococcus neoformans ccr4⌬ mutant lacking the major deadenylase involved in regulated mRNA decay was defective in host temperature adaptation and therefore virulence. In this study, the ccr4⌬ mutant was found to exhibit characteristics of chronic unfolded-protein response (UPR) engagement in both the gene expression profile and phenotype. We demonstrate that host temperature adaptation in C. neoformans is accompanied by transient induction of the endoplasmic reticulum (ER) stress response and that Ccr4-dependent posttranscriptional gene regulation contributes to resolution of ER stress during host temperature adaptation.The pathogenic fungus Cryptococcus neoformans is one of two species of cryptococci commonly associated with infection in humans (2, 6). Unifying characteristics of the pathogenic cryptococci include the production of a polysaccharide capsule, the ability to form melanin pigments through the activity of the multicopper oxidase laccase, and the ability to adapt to and thrive at mammalian host temperature. Adaptation of C. neoformans to the host temperature is accompanied by major changes in gene expression as measured by microarray analysis and serial analysis of gene expression (SAGE) (5,19,29). This modulation of gene expression likely requires alterations in mRNA synthesis rates through activation of transcriptional transactivators and repressors, as well as alterations in chromatin structure. In addition to mRNA synthesis, our previous studies of a C. neoformans ccr4⌬ mutant lacking the major mRNA deadenylase involved in regulated mRNA turnover suggest a role for posttranscriptional regulation of gene expression in C. neoformans host temperature adaptation (24).Destabilization of specific transcripts in response to stress is highly conserved. In the model yeast Saccharomyces cerevisiae, deletion of CCR4 results in stabilization of transcripts encoding distinct functional classes (ribosome biogenesis, translation initiation, and tRNA synthesis) in response to temperature stress (13). In mammalian cells, subsets of transcripts were destabilized in response to heat shock, and induction of endoplasmic reticulum (ER) stress by treatment with tunicamycin or potentiation of ER calcium release by thapsigargin treatment triggered destabilization of a subset of mRNAs in which are included several transcripts encoding ribosomal proteins (8). This suggests that in response to heat shock and ER stress, distinct pools of transcripts representing specific cellular processes are targeted for degradation.The conserved ER stress response involves engagement of the unfolded-protein response (UPR) and the ER-associated degradation (ERAD) pathway (18). The UPR serves to retool the ER for enhanced protein folding, and ERAD serves to remove unfolded proteins from the ER lumen and shunt them into a degradative pathway. Microarray analyses performed in S. cerevisiae ...

The mitochondrial genome of kinetoplastids, including species of Trypanosoma and Leishmania, is an unprecedented DNA structure of catenated maxicircles and minicircles. Maxicircles represent the typical mitochondrial genome encoding components of the respiratory complexes and ribosomes. However, most mRNA sequences are cryptic, and their maturation requires a unique U insertion/deletion RNA editing. Minicircles encode hundreds of small guide RNAs (gRNAs) that partially anneal with unedited mRNAs and direct the extensive editing. Trypanosoma brucei gRNAs and mRNAs are transcribed as polycistronic precursors, which undergo processing preceding editing; however, the relevant nucleases are unknown. We report the identification and functional characterization of a close homolog of editing endonucleases, mRPN1 (mitochondrial RNA precursor-processing endonuclease 1), which is involved in gRNA biogenesis. Recombinant mRPN1 is a dimeric dsRNAdependent endonuclease that requires Mg 2+ , a critical catalytic carboxylate, and generates 2-nucleotide 39 overhangs. The cleavage specificity of mRPN1 is reminiscent of bacterial RNase III and thus is fundamentally distinct from editing endonucleases, which target a single scissile bond just 59 of short duplexes. An inducible knockdown of mRPN1 in T. brucei results in loss of gRNA and accumulation of precursor transcripts (pre-gRNAs), consistent with a role of mRPN1 in processing. mRPN1 stably associates with three proteins previously identified in relatively large complexes that do not contain mRPN1, and have been linked with multiple aspects of mitochondrial RNA metabolism. One protein, TbRGG2, directly binds mRPN1 and is thought to modulate gRNA utilization by editing complexes. The proposed participation of mRPN1 in processing of polycistronic RNA and its specific protein interactions in gRNA expression are discussed.

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3=-to-5= progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. T rypanosome RNA editing entails the precise addition and removal of uridine nucleotides in mitochondrial RNAs. In Trypanosoma brucei, 12 of the 18 mitochondrially encoded mRNAs require editing for maturation prior to their translation. Essential players in this process are mitochondrially encoded 50-to 60-nucleotide (nt)-long guide RNAs (gRNAs), which direct the positions of uridine insertion and deletion through base-pairing interactions. The editing cycle is initiated upon association of a cognate gRNA with preedited mRNA by formation of a short anchor duplex. Editing catalysis is mediated by multiprotein complexes called editosomes or RNA editing core complexes (RECCs), and editing efficiency is achieved through the actions of transiently associating accessory factors (8,16,45,55,56,62,63). Following annealing of gRNA/preedited mRNA, a gRNAdirected endonuclease cleaves the premRNA at the site of gRNA/mRNA mismatch, and U insertion or deletion is catalyzed by terminal uridylyl transferase or U-specific exoribonuclease activities, respectively. The mRNA is then resealed by RNA ligase in preparation for a subsequent editing cycle. The editing cycles continue until gRNA/mRNA base pairing is extended along the entire length of the gRNA. gRNAs are then presumably exchanged, and the process continues, proceeding in a general 3=-to-5= direction along the mRNA. While "minimally edited mRNAs" are edited only in small regions, the majority of mRNAs are edited throughout their lengths and thus are termed panedited. Complete editing of panedited mRNAs requires sequential utilization of dozens of gRNAs.RNA-editing accessory factors are thought to coordinate recruitment of RNAs to the editosome, to direct correct gRNA/ mRNA annealing, and to regulate editing progression by modulating RNA-RNA and RNA-protein interactions. Accessory factors studied to date include RBP16, MRP1/2, and TbRGG2, all of which bind and anneal RNAs, T. brucei RGG1 (TbRGG1), and the RNA helicase REH1 (2,28,33,40,44,50,57,65). TbRGG2 is a component of a multiprotein complex, Mitochondrial RNA Binding Complex 1 (MRB1, also known as GRBC), which contains numerous proteins ...