Minimization and complete loss of general transcription factor proteins in the intracellular parasite Encephalitozoon cuniculi.

Genome compaction is a common evolutionary feature of parasites. The unicellular, obligate intracellular parasite Encephalitozoon cuniculi has one of smallest known eukaryotic genomes, and is nearly four times smaller than its distant fungi relative, the budding yeast Saccharomyces cerevisiae. Comparison of the proteins encoded by compacted genomes to those encoded by larger genomes can reveal the most highly conserved features of the encoded proteins. In this study, we identified the proteins comprising the RNA polymerases and their corresponding general transcription factors by using several bioinformatic approaches to compare the transcription machinery of E. cuniculi and S. cerevisiae. Surprisingly, our analyses revealed an overall reduction in the size of the proteins comprising transcription machinery of E. cuniculi, which includes the loss of entire regions or functional domains from proteins, as well as the loss of entire proteins and complexes. Unexpectedly, we found that the E. cuniculi ortholog of Rpc37 (a RNA Polymerase III subunit) more closely resembles the H. sapiens ortholog of Rpc37 than the S. cerevisiae ortholog of Rpc37, in both size and structure. Overall, our findings provide new insight into the minimal core eukaryotic transcription machinery and help define the most critical features of Pol components and general transcription factors.

Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits.

Eukaryotic DNA-dependent RNA polymerases (Pols I-III) encode two distinct alpha-like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H leukodystrophy, and primary ovarian sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a "humanized" yeast that we used to characterize the molecular consequence of the TCS-causingPOLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.

A clinically-relevant residue of POLR1D is required for Drosophila development.

BACKGROUND: POLR1D is a subunit of RNA Polymerases I and III, which synthesize ribosomal RNAs. Dysregulation of these polymerases cause several types of diseases, including ribosomopathies. The craniofacial disorder Treacher Collins Syndrome (TCS) is a ribosomopathy caused by mutations in several subunits of RNA Polymerase I, including POLR1D. Here, we characterized the effect of a missense mutation in POLR1D and RNAi knockdown of POLR1D on Drosophila development. RESULTS: We found that a missense mutation in Drosophila POLR1D (G30R) reduced larval rRNA levels, slowed larval growth, and arrested larval development. Remarkably, the G30R substitution is at an orthologous glycine in POLR1D that is mutated in a TCS patient (G52E). We showed that the G52E mutation in human POLR1D, and the comparable substitution (G30E) in Drosophila POLR1D, reduced their ability to heterodimerize with POLR1C in vitro. We also found that POLR1D is required early in the development of Drosophila neural cells. Furthermore, an RNAi screen revealed that POLR1D is also required for development of non-neural Drosophila cells, suggesting the possibility of defects in other cell types. CONCLUSIONS: These results establish a role for POLR1D in Drosophila development, and present Drosophila as an attractive model to evaluate the molecular defects of TCS mutations in POLR1D.

Molecular Topology of RNA Polymerase I Upstream Activation Factor.

Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.