The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans.

Methylation of carbon-5 of cytosines (m5 C) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m5 C-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of m5 C in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m5 C sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of m5 C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m5 C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m5 C34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of m5 C tRNA wobble methylation in the adaptation to higher temperatures.

Tissue- and sex-specific small RNAomes reveal sex differences in response to the environment.

RNA interference (RNAi) related pathways are essential for germline development and fertility in metazoa and can contribute to inter- and trans-generational inheritance. In the nematode Caenorhabditis elegans, environmental double-stranded RNA provided by feeding can lead to heritable changes in phenotype and gene expression. Notably, transmission efficiency differs between the male and female germline, yet the underlying mechanisms remain elusive. Here we use high-throughput sequencing of dissected gonads to quantify sex-specific endogenous piRNAs, miRNAs and siRNAs in the C. elegans germline and the somatic gonad. We identify genes with exceptionally high levels of secondary 22G RNAs that are associated with low mRNA expression, a signature compatible with silencing. We further demonstrate that contrary to the hermaphrodite germline, the male germline, but not male soma, is resistant to environmental RNAi triggers provided by feeding, in line with previous work. This sex-difference in silencing efficacy is associated with lower levels of gonadal RNAi amplification products. Moreover, this tissue- and sex-specific RNAi resistance is regulated by the germline, since mutant males with a feminized germline are RNAi sensitive. This study provides important sex- and tissue-specific expression data of miRNA, piRNA and siRNA as well as mechanistic insights into sex-differences of gene regulation in response to environmental cues.

SID-2 negatively regulates development likely independent of nutritional dsRNA uptake.

RNA interference (RNAi) is a gene regulatory mechanism based on RNA-RNA interaction conserved through eukaryotes. Surprisingly, many animals can take-up human-made double stranded RNA (dsRNA) from the environment to initiate RNAi suggesting a mechanism for dsRNA-based information exchange between organisms and their environment. However, no naturally occurring example has been identified since the discovery of the phenomenon 22 years ago. Therefore it remains enigmatic why animals are able to take up dsRNA. Here, we explore other possible functions by performing phenotypic studies of dsRNA uptake deficient sid-2 mutants in Caenorhabditis elegans. We find that SID-2 does not have a nutritional role in feeding experiments using genetic sensitized mutants. Furthermore, we use robot assisted imaging to show that sid-2 mutants accelerate growth rate and, by maternal contribution, body length at hatching. Finally, we perform transcriptome and lipidome analysis showing that sid-2 has no effect on energy storage lipids, but affects signalling lipids and the embryo transcriptome. Overall, these results suggest that sid-2 has mild effects on development and is unlikely functioning in the nutritional uptake of dsRNA. These findings broaden our understanding of the biological role of SID-2 and motivate studies identifying the role of environmental dsRNA uptake.

Artificial and natural RNA interactions between bacteria and C. elegans.

Nineteen years after Lisa Timmons and Andy Fire first described RNA transfer from bacteria to C. elegans in an experimental setting 48 the biologic role of this trans-kingdom RNA-based communication remains unknown. Here we summarize our current understanding on the mechanism and potential role of such social RNA.

Intergenerational adaptations to stress are evolutionarily conserved, stress-specific, and have deleterious trade-offs.

Despite reports of parental exposure to stress promoting physiological adaptations in progeny in diverse organisms, there remains considerable debate over the significance and evolutionary conservation of such multigenerational effects. Here, we investigate four independent models of intergenerational adaptations to stress in Caenorhabditis elegans - bacterial infection, eukaryotic infection, osmotic stress, and nutrient stress - across multiple species. We found that all four intergenerational physiological adaptations are conserved in at least one other species, that they are stress -specific, and that they have deleterious tradeoffs in mismatched environments. By profiling the effects of parental bacterial infection and osmotic stress exposure on progeny gene expression across species, we established a core set of 587 genes that exhibited a greater than twofold intergenerational change in expression in response to stress in C. elegans and at least one other species, as well as a set of 37 highly conserved genes that exhibited a greater than twofold intergenerational change in expression in all four species tested. Furthermore, we provide evidence suggesting that presumed adaptive and deleterious intergenerational effects are molecularly related at the gene expression level. Lastly, we found that none of the effects we detected of these stresses on C. elegans F1 progeny gene expression persisted transgenerationally three generations after stress exposure. We conclude that intergenerational responses to stress play a substantial and evolutionarily conserved role in regulating animal physiology and that the vast majority of the effects of parental stress on progeny gene expression are reversible and not maintained transgenerationally.

DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans.

Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.

Time-dependent distribution of sulphur, sulphate and glutathione in wheat tissues and grain as affected by three sulphur fertilization levels and late S fertilization.

Sulphur (S) fertilization has beneficial effects on yield and protein composition of mature wheat kernels. However, to understand the impact of S fertilization on storage protein composition, synthesis of S-containing compounds and their distribution during grain development has to be examined. A pot experiment with Triticum aestivum cultivar Türkis under three S fertilization levels (0 g, 0.1 g und 0.2 g S per pot) and a late S fertilization level at ear emergence was carried out. Stalk and leaves, flag leaves, ears and kernels were harvested separately during grain development at ear emergence, milk ripeness and maturity, and analyzed for elemental S, sulphate, glutathione, and protein concentration. Sulphate is the major S compound in stalk, leaf and ears at the start of grain development, whereas glutathione is more important for synthesis of S-containing proteins in the grain. The discrepancy of S concentration comparing low and high S fertilization became obvious after milk ripeness. The N/S ratios in ears at ear emergence and milk ripeness reflected the later N/S ratio in mature grain. Late S fertilization increased sulphate concentrations in the flag leaf within a short period of about two weeks at ear emergence. Late S fertilization prevented S deficiency in late stages of wheat growth and further enabled equal concentrations of S, glutathione and protein in all wheat organs compared to an S application only at sowing.