Characterization of Tau95 led to the identification of a four-subunit TFIIIC complex in trypanosomatid parasites.

RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB, and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first published draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131, and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major. KEY POINTS: • A four-subunit TFIIIC complex is present in T. brucei and L. major • TbTau95 associates with tRNA and U2 snRNA genes • Putative interacting partners of Tau95 might include some RNAP II regulators.

Mitochondrial Metabolism in the Spotlight: Maintaining Balanced RNAP III Activity Ensures Cellular Homeostasis.

RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the S. cerevisiae maf1Δ strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains maf1Δ (with increased tRNA abundance) and rpc128-1007 (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on 13C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. rpc128-1007, the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell's intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types.

Identification, validation, and characterization of noncanonical miRNAs.

Many eukaryotes and some viruses encode microRNAs (miRNAs), small RNAs that post-transcriptionally regulate gene expression. While most miRNAs are generated through the activity of RNA Polymerase II (RNAP II) and subsequent processing by Drosha and Dicer, some viral miRNAs utilize alternative pathways of biogenesis. Some members of the herpesvirus and retrovirus families can direct synthesis of miRNAs through RNAP III transcription rather than RNAP II and can utilize atypical enzymes to generate miRNAs. Though the advantages of alternative miRNA biogenesis remain unclear for herpesviruses, the retroviral miRNA biogenesis routes allow the RNAP II transcribed retroviral genome to escape Drosha cleavage while still expressing abundant, biologically-active miRNAs. These RNAP III-derived miRNAs have unique characteristics that allow for their identification and characterization. In this article, we describe procedures to predict, validate, and characterize RNAP III-transcribed miRNAs and other small RNAs, while providing resources that are also useful for canonical miRNAs.