Ageing-associated changes in transcriptional elongation influence longevity.

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.

Functional characterization and transcriptomic profiling of a spheroid-forming midgut cell line from Helicoverpa zea (Lepidoptera: Noctuidae).

Insect cell lines have been frequently used in insect science research in recent years. Establishment of cell lines from specialized tissues like the lepidopteran midgut is expected to facilitate research efforts towards the understanding of uptake and metabolic properties, as well as the design of assays for use in pesticide discovery. However, the number of available lines from specialized tissues of insects and the level of understanding of the biological processes taking place in insect cells is far behind mammalian systems. In this study we examine two established cell lines of insect midgut origin, investigate their growth parameters and amenability to transfection and genetic manipulation, and test their potential to form spheroid-like 3D structures. Our results indicate that a midgut-derived cell line from Helicoverpa zea, RP-HzGUT-AW1, is amenable to genetic manipulation by transfection with a standard insect expression vector and has excellent ability to form spheroids. To further investigate the differentiation status of this line, we examined for expression of several candidate marker genes from different midgut cell types, enterocytes (ECs), Goblet cells (GCs), enteroendocrine cells (EEs) and intestinal stem cells (ISCs), indicating that both certain ISC and certain differentiated cell markers were present. To acquire a more detailed perspective of the differentiation landscape of the specific cells, we performed an RNAseq analysis of RP-HzGUT-AW1 grown either in 2D or 3D cultures. We hypothesize that RP-HzGUT-AW1 are in an "arrested" developmental stage between ISC and terminal differentiation. Furthermore, an enrichment of stress response and oxidoreductase genes was observed in the spheroid samples while no significant difference was evident in differentiation markers between cells grown in 2D and 3D. These results render RP-HzGUT-AW1 as the most well-characterized insect gut derived cell line so far, and lay the groundwork for future work investigating midgut cell lines application potential.

Untangling a Gordian knot: the role of a GluCl3 I321T mutation in abamectin resistance in Tetranychus urticae.

BACKGROUND: The cys-loop ligand-gated ion channels, including the glutamate-gated chloride channel (GluCl) and GABA-gated chloride channel (Rdl) are important targets for drugs and pesticides. The macrocyclic lactone abamectin primarily targets GluCl and is commonly used to control the spider mite Tetranychus urticae, an economically important crop pest. However, abamectin resistance has been reported for multiple T. urticae populations worldwide, and in several cases was associated with the mutations G314D in GluCl1 and G326E in GluCl3. Recently, an additional I321T mutation in GluCl3 was identified in several abamectin resistant T. urticae field populations. Here, we aim to functionally validate this mutation and determine its phenotypic strength. RESULTS: The GluCl3 I321T mutation was introgressed into a T. urticae susceptible background by marker-assisted backcrossing, revealing contrasting results in phenotypic strength, ranging from almost none to 50-fold. Next, we used CRISPR-Cas9 to introduce I321T, G314D and G326E in the orthologous Drosophila GluCl. Genome modified flies expressing GluCl I321T were threefold less susceptible to abamectin, while CRISPRed GluCl G314D and G326E flies were lethal. Last, functional analysis in Xenopus oocytes revealed that the I321T mutation might reduce GluCl3 sensitivity to abamectin, but also suggested that all three T. urticae Rdls are affected by abamectin. CONCLUSION: Three different techniques were used to characterize the role of I321T in GluCl3 in abamectin resistance and, combining all results, our analysis suggests that the I321T mutation has a complex role in abamectin resistance. Given the reported subtle effect, additional synergistic factors in resistance warrant more investigation. © 2020 Society of Chemical Industry.