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The role of many enzymes extends beyond their dedicated catalytic activity by fulfilling important cellular functions in a catalysis-independent fashion. In this aspect, little is known about 3′-end RNA-modifying enzymes that belong to the class of nucleotidyl transferases. Among these are noncanonical poly(A) polymerases, a group of evolutionarily conserved enzymes that are critical for gene expression regulation, by adding adenosines to the 3′-end of RNA targets. In this study, we investigate whether the functions of the cytoplasmic poly(A) polymerase (cytoPAP) GLD-2 in C. elegans germ cells exclusively depend on its catalytic activity. To this end, we analyzed a specific missense mutation affecting a conserved amino acid in the catalytic region of GLD-2 cytoPAP. Although this mutated protein is expressed to wild-type levels and incorporated into cytoPAP complexes, we found that it cannot elongate mRNA poly(A) tails efficiently or promote GLD-2 target mRNA abundance. Furthermore, germ cell defects in animals expressing this mutant protein strongly resemble those lacking the GLD-2 protein altogether, arguing that only the polyadenylation activity of GLD-2 is essential for gametogenesis. In summary, we propose that all known molecular and biological functions of GLD-2 depend on its enzymatic activity, demonstrating that polyadenylation is the key mechanism of GLD-2 functionality. Our findings highlight the enzymatic importance of noncanonical poly(A) polymerases and emphasize the pivotal role of poly(A) tail-centered cytoplasmic mRNA regulation in germ cell biology.

RNA-binding proteins (RBPs) are important regulators of gene expression programs, especially during gametogenesis. How the abundance of particular RBPs is restricted to defined stages of meiosis remains largely elusive. Here, we report a molecular pathway that subjects two nonrelated but broadly evolutionarily conserved translational regulators (CPB-3/CPEB and GLD-1/STAR) to proteosomal degradation in Caenorhabditis elegans germ cells at the transition from pachytene to diplotene of meiotic prophase. Both RBPs are recognized by the same ubiquitin ligase complex, containing the molecular scaffold Cullin-1 and the tumor suppressor SEL-10/FBXW7 as its substrate recognition subunit. Destabilization of either RBP through this Skp, Cullin, F-box-containing complex (SCF) ubiquitin ligase appears to loosen its negative control over established target mRNAs, and presumably depends on a prior phosphorylation of CPB-3 and GLD-1 by MAPK (MPK-1), whose activity increases in mid- to late pachytene to promote meiotic progression and oocyte differentiation. Thus, we propose that the orchestrated degradation of RBPs via MAPK-signaling cascades during germ cell development may act to synchronize meiotic with sexual differentiation gene expression changes.

The role of many enzymes extends beyond their dedicated catalytic activity by fulfilling important cellular functions in a catalysis-independent fashion. In this aspect, little is known about 3′-end RNA-modifying enzymes that belong to the class of nucleotidyl transferases. Among these are noncanonical poly(A) polymerases, a group of evolutionarily conserved enzymes that are critical for gene expression regulation, by adding adenosines to the 3′-end of RNA targets. In this study, we investigate whether the functions of the cytoplasmic poly(A) polymerase (cytoPAP) GLD-2 in C. elegans germ cells exclusively depend on its catalytic activity. To this end, we analyzed a specific missense mutation affecting a conserved amino acid in the catalytic region of GLD-2 cytoPAP. Although this mutated protein is expressed to wild-type levels and incorporated into cytoPAP complexes, we found that it cannot elongate mRNA poly(A) tails efficiently or promote GLD-2 target mRNA abundance. Furthermore, germ cell defects in animals expressing this mutant protein strongly resemble those lacking the GLD-2 protein altogether, arguing that only the polyadenylation activity of GLD-2 is essential for gametogenesis. In summary, we propose that all known molecular and biological functions of GLD-2 depend on its enzymatic activity, demonstrating that polyadenylation is the key mechanism of GLD-2 functionality. Our findings highlight the enzymatic importance of noncanonical poly(A) polymerases and emphasize the pivotal role of poly(A) tail-centered cytoplasmic mRNA regulation in germ cell biology.

Poly(A) tail length is a readout of an mRNA’s translatability and stability, especially in developmental systems. PolyAdenylation Test (PAT) assays attempt to quickly measure the average poly(A) tail length of RNAs of experimental interest. Here we present sPAT, splint-mediated PAT, a procedure that uses a DNA splint to aid in the ligation of an RNA-tag to the poly(A) tail of an mRNA. In comparison to other PAT methodologies, including ePAT, sPAT is highly sensitive to low-abundance mRNAs, gives a more accurate profile of the poly(A) tail distribution, and requires little starting material. To demonstrate its strength, we calibrated sPAT on defined poly(A) tails of synthetic mRNAs, reassessed developmentally regulated poly(A) tail-length changes of known mRNAs from established model organisms, and extended it to the emerging evolutionary developmental nematode model Pristionchus pacificus. Lastly, we used sPAT to analyze the contribution of the two cytoplasmic poly(A) polymerases GLD-2 and GLD-4, and the deadenylase CCR-4, onto Caenorhabditis elegans gld-1 mRNA that encodes a translationally controlled tumor suppressor whose poly(A) tail length measurement proved elusive.

To survive in Antarctica, early explorers of Antarctica's Heroic Age erected wooden buildings and brought in large quantities of supplies. The introduction of wood and other organic materials may have provided new nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials. From 30 samples taken fromDiscoveryHut, 156 filamentous fungi were isolated on selective media. Of these, 108 were screened for hydrolytic activity on carboxymethyl cellulose, of which 29 demonstrated activities. Endo-1, 4-β-glucanase activity was confirmed in the extracellular supernatant from seven isolates when grown at 4°C, and also when they were grown at 15°C.Cladosporium oxysporumandGeomycessp. were shown to grow on a variety of synthetic cellulose substrates and to use cellulose as a nutrient source at temperate and cold temperatures. The research findings from the present study demonstrate that Antarctic filamentous fungi isolated from a variety of substrates (wood, straw, and food stuffs) are capable of cellulose degradation and can grow well at low temperatures.

Pristionchus pacificus has been developed as a nematode satellite organism in evolutionary developmental biology. Detailed studies of vulva development revealed multiple differences in genetic and molecular control in P. pacificus compared to the model organism Caenorhabditis elegans. To place evolutionary developmental biology in a comprehensive evolutionary context, such studies have to be complemented with ecology. In recent field studies in western Europe and eastern North America we found 11 Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. However, P. pacificus was not commonly found in association with scarab beetles in these studies. Here, we describe the results of a similar survey of scarab beetles in Japan. Pristionchus pacificus was the most common Pristionchus species on scarab beetles in Japan, with 40 out of 43 (93%) isolates. The other Pristionchus isolates represent three novel species, which we refer to as Pristionchus sp. 11, Pristionchus sp. 14, and Pristionchus sp. 15. Thirty-seven of the established P. pacificus strains were found on the oriental beetle Exomala orientalis. Laboratory studies with the sex pheromone (Z)-7-tetradecen-2-one of the oriental beetle revealed that P. pacificus shows strong olfactory attraction to the beetle's sex pheromone, which provides a potential mechanism for the recognition and interaction of P. pacificus and E. orientalis. Together, this study identifies P. pacificus as the most common Pristionchus nematode in field studies in Japan, identifies E. orientalis as an important host species, and provides the basis for the ecology of P. pacificus.

While canonical 3′end modifications of mRNAs or tRNAs are well established, recent technological advances in RNA analysis have given us a glimpse of how widespread other types of distinctive 3′end modifications appear to be. Next to alternative nuclear or cytoplasmic polyadenylation mechanisms, evidence accumulated for a variety of 3′end mono-nucleotide and oligo-nucleotide additions of primarily adenosines or uracils on a variety of RNA species. Enzymes responsible for such non-templated additions are non-canonical RNA nucleotidyltransferases, which possess surprising flexibility in RNA substrate selection and enzymatic activity. We will highlight recent findings supporting the view that RNA nucleotidyltransferase activity, RNA target selection and sub-compartimentalization are spatially, temporally and physiologically regulated by dedicated co-factors. Along with the diversification of non-coding RNA classes, the evolutionary conservation of these multifaceted RNA modifiers underscores the prevalence and importance of diverse 3′end formation mechanisms.

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50-80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo.