Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans

Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.

The Role of pkc-3 and Genetic Suppressors in Caenorhabditis elegans Epithelial Cell Junction Formation

Epithelial cells form intercellular junctions to strengthen cell–cell adhesion and limit diffusion, allowing epithelia to function as dynamic tissues and barriers separating internal and external environments. Junctions form as epithelial cells differentiate; clusters of junction proteins first concentrate apically, then mature into continuous junctional belts that encircle and connect each cell. In mammals and Drosophila, atypical protein kinase C (aPKC) is required for junction maturation, although how it contributes to this process is poorly understood. A role for the Caenorhabditis elegans aPKC homolog PKC-3 in junction formation has not been described previously. Here, we show that PKC-3 is essential for junction maturation as epithelia first differentiate. Using a temperature-sensitive allele of pkc-3 that causes junction breaks in the spermatheca and leads to sterility, we identify intragenic and extragenic suppressors that render pkc-3 mutants fertile. Intragenic suppressors include an unanticipated stop-to-stop mutation in the pkc-3 gene, providing evidence for the importance of stop codon identity in gene activity. One extragenic pkc-3 suppressor is a loss-of-function allele of the lethal(2) giant larvae homolog lgl-1, which antagonizes aPKC within epithelia of Drosophila and mammals, but was not known previously to function in C. elegans epithelia. Finally, two extragenic suppressors are loss-of-function alleles of sups-1—a previously uncharacterized gene. We show that SUPS-1 is an apical extracellular matrix protein expressed in epidermal cells, suggesting that it nonautonomously regulates junction formation in the spermatheca. These findings establish a foundation for dissecting the role of PKC-3 and interacting genes in epithelial junction maturation.

Suppressor screening reveals common kleisin–hinge interaction in condensin and cohesin, but different modes of regulation

Cohesin and condensin play fundamental roles in sister chromatid cohesion and chromosome segregation, respectively. Both consist of heterodimeric structural maintenance of chromosomes (SMC) subunits, which possess a head (containing ATPase) and a hinge, intervened by long coiled coils. Non-SMC subunits (Cnd1, Cnd2, and Cnd3 for condensin; Rad21, Psc3, and Mis4 for cohesin) bind to the SMC heads. Here, we report a large number of spontaneous extragenic suppressors for fission yeast condensin and cohesin mutants, and their sites were determined by whole-genome sequencing. Mutants of condensin’s non-SMC subunits were rescued by impairing the SUMOylation pathway. Indeed, SUMOylation of Cnd2, Cnd3, and Cut3 occurs in midmitosis, and Cnd3 K870 SUMOylation functionally opposes Cnd subunits. In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1-mediated chromatin remodeling. In addition, distinct regulations were discovered for condensin and cohesin hinge mutants. Mutations in the N-terminal helix bundle [containing a helix–turn–helix (HTH) motif] of kleisin subunits (Cnd2 and Rad21) rescue virtually identical hinge interface mutations in cohesin and condensin, respectively. These mutations may regulate kleisin’s interaction with the coiled coil at the SMC head, thereby revealing a common, but previously unknown, suppression mechanism between the hinge and the kleisin N domain, which is required for successful chromosome segregation. We propose that in both condensin and cohesin, the head (or kleisin) and hinge may interact and collaboratively regulate the resulting coiled coils to hold and release chromosomal DNAs.

Suppressor mutation analysis combined with 3D modeling explains cohesin’s capacity to hold and release DNA

Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe. Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head–Rad21, or Psm3 coiled coil–Rad21. Molecular-dynamics analysis indicates that the intermolecular β-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the “hold-and-release” model, which may be considered as an alternative to the prevailing “ring” model.

Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

A Gripping Tale of Ribosomal Frameshifting: Extragenic Suppressors of Frameshift Mutations Spotlight P-Site Realignment

SUMMARYMutants of translation components which compensate for both −1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook “yardstick” model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the −1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3′ CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1α to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

Assembly of the MexAB-OprM Multidrug Pump of Pseudomonas aeruginosa: Component Interactions Defined by the Study of Pump Mutant Suppressors

ABSTRACT In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the α/β domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic α-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.

The STE20/Germinal Center Kinase POD6 Interacts with the NDR Kinase COT1 and Is Involved in Polar Tip Extension inNeurospora crassa

Members of the Ste20 and NDR protein kinase families are important for normal cell differentiation and morphogenesis in various organisms. We characterized POD6 (NCU02537.2), a novel member of the GCK family of Ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in the filamentous fungus Neurospora crassa. pod-6 and the NDR kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered cell-wall composition. We suggest that POD6 and COT1 act in the same genetic pathway, based on the fact that both pod-6 and cot-1 can be suppressed by 1) environmental stresses, 2) altering protein kinase A activity, and 3) common extragenic suppressors (ropy, as well as gul-1, which is characterized here as the ortholog of the budding and fission yeasts SSD1 and Sts5, respectively). Unlinked noncomplementation of cot-1/pod-6 alleles indicates a potential physical interaction between the two kinases, which is further supported by coimmunoprecipitation analyses, partial colocalization of both proteins in wild-type cells, and their common mislocalization in dynein/kinesin mutants. We conclude that POD6 acts together with COT1 and is essential for polar cell extension in a kinesin/dynein-dependent manner in N. crassa.