dourine ͉ mitochondria ͉ protozoa ͉ RNA editing ͉ surra

The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in Trypanosoma brucei. Based on the phenotype caused by down-regulation of these two proteins, it was proposed to play an unspecified role in RNA editing. RNAi silencing of three newly characterized protein subunits, guide RNA associated proteins (GAPs) 1 and 2 as well as a predicted DExD/H-box RNA helicase, show they are essential for cell growth in the procyclic stage. Furthermore, their down-regulation leads to inhibition of editing in only those mRNAs for which minicircleencoded guide (g) RNAs are required. However, editing remains unaffected when the maxicircle-encoded cis-acting gRNA is employed. Interestingly, all three proteins are necessary for the expression of the minicircle-encoded gRNAs. Moreover, downregulation of a fourth assayed putative MRB1 subunit, Nudix hydrolase, does not appear to destabilize gRNAs, and downregulation of this protein has a general impact on the stability of maxicircle-encoded RNAs. GAP1 and 2 are also essential for the survival of the bloodstream stage, in which the gRNAs become eliminated upon depletion of either protein.Immunolocalization revealed that GAP1 and 2 are concentrated into discrete spots along the mitochondrion, usually localized in the proximity of the kinetoplast. Finally, we demonstrate that the same mtRNA polymerase known to transcribe the maxicircle mRNAs may also have a role in expression of the minicircle-encoded gRNAs.

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.

RNA editing, which adds sequence information to RNAs posttranscriptionally, is a widespread phenomenon throughout eukaryotes. The most complex form of this process is the uridine (U) insertion/deletion editing that occurs in the mitochondria of kinetoplastid protists. RNA editing in these flagellates is specified by trans acting guide RNAs and entails the insertion of hundreds and deletion of dozens of U residues from mitochondrial RNAs to produce mature, translatable mRNAs. An emerging model indicates that the machinery required for trypanosome RNA editing is much more complicated than previously appreciated. A family of RNA editing core complexes (RECCs), which contain the required enzymes and several structural proteins, catalyze cycles of U insertion and deletion. A second, dynamic multi-protein complex, the Mitochondrial RNA Binding 1 (MRB1) complex, has recently come to light as another essential component of the trypanosome RNA editing machinery. MRB1 likely serves as the platform for kinetoplastid RNA editing, and plays critical roles in RNA utilization and editing processivity. MRB1 also appears to act as a hub for coordination of RNA editing with additional mitochondrial RNA processing events. This review highlights the current knowledge regarding the complex molecular machinery involved in trypanosome RNA editing.

Kinetoplastids are flagellated protozoans, whose members include the pathogens Trypanosoma brucei, T. cruzi and Leishmania species, that are considered among the earliest diverging eukaryotes with a mitochondrion. This organelle has become famous because of its many unusual properties, which are unique to the order Kinetoplastida, including an extensive kinetoplast DNA network and U-insertion/deletion type RNA editing of its mitochondrial transcripts. In the last decade, considerable progress has been made in elucidating the complex machinery of RNA editing. Moreover, our understanding of the structure and replication of kinetoplast DNA has also dramatically improved. Much less however, is known, about the developmental regulation of RNA editing, its integration with other RNA maturation processes, stability of mitochondrial mRNAs, or evolution of the editing process itself. Yet the profusion of genomic data recently made available by sequencing consortia, in combination with methods of reverse genetics, hold promise in understanding the complexity of this exciting organelle, knowledge of which may enable us to fight these often medically important protozoans.

The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1).

Trypanosomes and leishmanias are widely known parasites of humans. However, they are just two out of several phylogenetic lineages that constitute the family Trypanosomatidae. Although dixeny - the ability to infect two hosts - is a derived trait of vertebrate-infecting parasites, the majority of trypanosomatids are monoxenous. Like their common ancestor, the monoxenous Trypanosomatidae are mostly parasites or commensals of insects. This review covers recent advances in the study of insect trypanosomatids, highlighting their diversity as well as genetic, morphological and biochemical complexity, which, until recently, was underappreciated. The investigation of insect trypanosomatids is providing an important foundation for understanding the origin and evolution of parasitism, including colonization of vertebrates and the appearance of human pathogens.

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.