Biology subjects

Hayman, D. J.

Publications and source records attributed to Hayman, D. J..

4 recordsLinked to original sources

bioRxiv2025

Knockdown of the fly spliceosome component Rbp1(orthologue of SRSF1) extends lifespan

Biological regulation is a highly intricate process and involves many layers of complexity even at the RNA level. Alternative splicing is crucial in the regulation of which components of a protein-coding gene are spliced into a translatable mRNA. During ageing splicing becomes dysregulated and alternative splicing has been shown to be involved in disease and known anti-aging treatments such as dietary restriction (DR) and mTOR suppression. In prior work we have shown that DR and mTOR suppression modulate the expression of the spliceosome in the fly (Drosophila melanogaster). Here, we manipulated the five top genes that change in expression in both these treatments. We found that knockdown (using conditional in vivo RNAi in adults) of some spliceosome components rapidly induce mortality, whereas one, Rbp1, extends lifespan. Treatments that have more instant benefits on longevity are more translatable. We therefore subsequently repeated the Rbp1 experiment, but initiating Rbp1 at later stages in adult life. We find that irrespective of age of induction, knockdown of Rbp1 extends lifespan. Our results posit the spliceosome itself as a hub of regulation that when targeted can extend lifespan, rendering it a promising target for geroscience.

physiology↗

bioRxiv2025

Suppression rather than activation of the integrated-stress-response (GCN2-ATF4) pathway extends lifespan in the fly

Stress response pathways are emerging as conserved modulators of lifespan. The prevailing hypothesis is that activation of stress responsive pathways, including the amino acid deprivation arm of the integrated stress response (ISR; the GCN2-ATF4 pathway) is pro-longevity. Activation of ATF4 orthologs extends lifespan in Saccharomyces cerevisiae (yeast) and Caenorhabditis elegans, but its role in other longer-lived organisms remains unclear. We comprehensively tested the role of the GCN2-ATF4 pathway in the fly (Drosophila melanogaster) for the first time. We used conditional genetic manipulation of dGCN2 and its downstream effector Drosophila ATF4 (crc; dATF4). In contrast to previous studies, we show that overexpression of dGCN2 and dATF4 significantly reduces lifespan, while knockdown (in vivo RNAi) of dATF4 extends lifespan. We confirmed dATF4 activity was successfully modulated using a fluorescent dATF4 activation reporter. Borrelidin, a tRNA synthetase inhibitor, significantly reduced lifespan in a both dATF4 and diet-dependent manner, independent of microbial load, showing our modulation of dATF4 altered nutrient to ISR signalling. We further conducted long-read RNA sequencing and found that our manipulation of dATF4 changed global transcription in opposite directions, including known ATF4 target genes. Enrichment analysis revealed that dATF4 overexpression may drive metabolic stress, while dATF4 knockdown upregulates proteostasis and DNA repair pathways. Our work reveals that ATF4 exhibits a dual, dose- and context-dependent role in ageing. Chronic dATF4 activation is detrimental in flies, while chronic suppression is pro-longevity. The GCN2-ATF4 pathway thus qualifies as a modifiable control of lifespan with cross-species relevance.

physiology↗

bioRxiv2024

Expansion of Drosophila haemocytes using a conditional GeneSwitch driver affects larval haemocyte function, but does not modulate adult lifespan or survival from infection

Macrophages are responsible for diverse and fundamental functions in vertebrates. Fruit flies harbour an innate immune system of which the most populous blood cell (haemocyte) type bears striking homology to the vertebrate macrophage. The importance of these cells has been demonstrated previously, where immune and developmental phenotypes have been observed upon haemocyte ablation using pro-apoptotic transgenes driven by the Hml promoter. Here we show that, as well as ablating Hml-positive cells in vivo using the pro-apoptotic transgene bax, we can also increase Hml-positive cell numbers using a constitutively-active form of ras. However, in adults, compared to larvae, total blood cell numbers were not significantly affected by experimental expansion or ablation. This therefore implies the existence of feedback mechanisms which regulate the number of haemocytes. No effect on lifespan was observed from driving ras and bax in Hml-positive cells using a conditional genetic system (Hml-GeneSwitch). Using a constitutive driver system, we did observe differences in lifespan, however we attribute this to differences in genetic background that could have led to spurious conclusions. Additionally, no effect of either transgene was observed upon infection with two different bacterial species, although a striking pupal lethality phenotype was observed upon expansion of Hml-positive cells in the context of a self-encapsulation mutant genetic background. The latter confirms that the change in Hml-positive cell number does result in a phenotype. The lack of adult phenotypes could be due to the strength of our experimental manipulation or due to compensation via feedback mechanisms that operate to maintain total blood cell numbers. Our study demonstrates the importance of a conditional approach to modulate haemocyte cell numbers in vivo which allows for more precise study of innate immune system function. This approach could be especially fruitful to uncover the mechanisms that regulate total blood cell numbers across development and ageing.

developmental biology↗

bioRxiv2023

miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity

microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR- 324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

cell biology↗