Mutation of RNA Pol III Subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and Disrupts Zebrafish Digestive Development

The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-Δ yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function.     

Coupling of RNA polymerase III assembly to cell cycle progression in Saccharomyces cerevisiae

Assembly of the RNA polymerases in both yeast and humans is proposed to occur in the cytoplasm prior to their nuclear import. Our previous studies identified a cold-sensitive mutation, rpc128-1007, in the yeast gene encoding the second largest Pol III subunit, Rpc128. rpc128-1007 is associated with defective assembly of Pol III complex and, in consequence, decreased level of tRNA synthesis. Here, we show that rpc128-1007 mutant cells remain largely unbudded and larger than wild type cells. Flow cytometry revealed that most rpc128-1007 mutant cells have G1 DNA content, suggesting that this mutation causes pronounced cell cycle delay in the G1 phase. Increased expression of gene encoding Rbs1, the Pol III assembly/import factor, could counteract G1 arrest observed in the rpc128-1007 mutant and restore wild type morphology of mutant cells. Concomitantly, cells lacking Rbs1 show a mild delay in G1 phase exit, indicating that Rbs1 is required for timely cell cycle progression. Using the double rpc128-1007 maf1Δ mutant in which tRNA synthesis is recovered, we confirmed that the Pol III assembly defect associated with rpc128-1007 is a primary cause of cell cycle arrest. Together our results indicate that impairment of Pol III complex assembly is coupled to cell cycle inhibition in the G1 phase.     

Molecular cloning and characteristics of rpc19+ and rpc40+ Schizosaccharomyces pombe genes, coding for common subunits of nuclear RNA polymerase I and III

Full-length copies of cDNAs of the rpc19+ and rpc40+ genes encoding the common subunits of nuclear RNA polymerases I and III and the corresponding fragments of chromosomes were isolated from genomic and cDNA libraries of Schizosaccharomyces pombe and characterized. It was established that the cloned genes are located on chromosomes III and II of the fission yeast, respectively. The rpc40+ gene lacks introns, and the rpc19+ gene contains two intervening sequences. The comparison of subunits Rpc19 (125 aa; M 13 722 Da; pI 4.51) and Rpc40 (348 aa; M 39 141 Da; pI 5.40) of Sz. pombe, whose characteristics were deduced from the sequences of their cDNAs, with the orthologous components of other eukaryotes allowed the most conserved structure-functional domains of these proteins to be identified.     

A genetic look at the active site of RNA polymerase III

rpc160-112, a mutant of the RNA polymerase III active site, is corrected in vivo by six second-site mutants obtained by random mutagenesis. These mutants introduce single-site amino acid replacements at the two large subunits of the enzyme. The mutated motifs are conserved in RNA polymerases I and II and, for some of them, in the bacterial enzyme, thus delineating key elements of the active site in eukaryotic RNA polymerases.     

Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair

Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair.     

A 14-kDa Arabidopsis thaliana RNA polymerase III subunit contains two α-motifs flanked by a highly charged C terminus

We have sequenced a cDNA and a gene, AtRPC14, from Arabidopsis thaliana (At) (ecotype Columbia) that encode a protein related to the yeast RNA polymerases (Pol) I and III subunits, yAC19. Polyclonal antibodies raised against the recombinant At polypeptide (AtC14) bind to the Pol I and/or III subunits of about 13–15 kDa, but do not bind to any Pol II subunit in Pol purified from cauliflower, wheat or At. The amino acid (aa) sequence derived from the AtRPC14 cDNA and genomic clones consists of 122 aa, as compared to the 142 aa in the yeast yAC19 subunit and 143 aa in a putative Caenorhabditis elegans CeAC16 subunit. AtC14, yAC19 and CeAC16 contain a conserved sequence of about 85 aa which is related to two motifs in the α subunit of Escherichia coli (Ec) Pol. AtC14 lacks a highly charged N terminus of about 50 aa found in both yAC19 and CeAC16, but has a highly charged C terminus of about 30 aa not found in yAC19 and CeAC16.     

Isolation and characterization of temperature-sensitive mutations in the gene (rpb3 ) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the α subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β′ subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, as in the case of the prokaryotic α₂β complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A–D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by over-expression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered.     

Mutator suppression and escape from replication error-induced extinction in yeast

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.