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.

Catalytic activity of the Bin3/MePCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster.

Methylphosphate Capping Enzyme (MePCE) monomethylates the gamma phosphate at the 5' end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MePCE in vitro, little is known about its functions in vivo, or what roles-if any-there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MePCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MePCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MePCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3Y795A) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3ΔMSM). Bin3ΔMSM mutant flies exhibited some-but not all-bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.

Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster.

Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5' end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MEPCE in vitro, little is known about its functions in vivo, or what roles- if any-there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MEPCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MEPCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MEPCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3Y795A) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3ΔMSM). Bin3ΔMSM mutant flies exhibited some-but not all-bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.

Coevolution of the Ess1-CTD axis in polar fungi suggests a role for phase separation in cold tolerance.

Most of the world's biodiversity lives in cold (-2° to 4°C) and hypersaline environments. To understand how cells adapt to such conditions, we isolated two key components of the transcription machinery from fungal species that live in extreme polar environments: the Ess1 prolyl isomerase and its target, the carboxy-terminal domain (CTD) of RNA polymerase II. Polar Ess1 enzymes are conserved and functional in the model yeast, Saccharomyces cerevisiae. By contrast, polar CTDs diverge from the consensus (YSPTSPS)26 and are not fully functional in S. cerevisiae. These CTDs retain the critical Ess1 Ser-Pro target motifs, but substitutions at Y1, T4, and S7 profoundly affected their ability to undergo phase separation in vitro and localize in vivo. We propose that environmentally tuned phase separation by the CTD and other intrinsically disordered regions plays an adaptive role in cold tolerance by concentrating enzymes and substrates to overcome energetic barriers to metabolic activity.

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.

Sequential Regulation of Maternal mRNAs through a Conserved cis-Acting Element in Their 3' UTRs.

Maternal mRNAs synthesized during oogenesis initiate the development of future generations. Some maternal mRNAs are either somatic or germline determinants and must be translationally repressed until embryogenesis. However, the translational repressors themselves are temporally regulated. We used polar granule component (pgc), a Drosophila maternal mRNA, to ask how maternal transcripts are repressed while the regulatory landscape is shifting. pgc, a germline determinant, is translationally regulated throughout oogenesis. We find that different conserved RNA-binding proteins bind a 10-nt sequence in the 3' UTR of pgc mRNA to continuously repress translation at different stages of oogenesis. Pumilio binds to this sequence in undifferentiated and early-differentiating oocytes to block Pgc translation. After differentiation, Bruno levels increase, allowing Bruno to bind the same sequence and take over translational repression of pgc mRNA. We have identified a class of maternal mRNAs that are regulated similarly, including zelda, the activator of the zygotic genome.

Paralog-Specific Functions of RPL7A and RPL7B Mediated by Ribosomal Protein or snoRNA Dosage in Saccharomyces cerevisiae.

Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.

Host co-factors of the retrovirus-like transposon Ty1.

BACKGROUND: Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. RESULTS: To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. CONCLUSION: Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 cDNA include characterized RNA metabolism and modification genes, consistent with their having roles in early steps in retrotransposition such as expression, nuclear export, translation, localization, or packaging of Ty1 RNA. Our results suggest that Bud21, Hcr1, Loc1, and Puf6 promote efficient synthesis or stability of Ty1 Gag.