Biology subjects

Mollet, I. G.

Publications and source records attributed to Mollet, I. G..

3 recordsLinked to original sources

bioRxiv2023

Transcriptome-wide analysis of primary human endothelial cell responses to 1 hour of protein translation inhibition identify nonsense mediated decay targets and a non-coding SLC11A2 exon as an acute biomarker

Nonsense mediated decay (NMD) lowers the cellular concentration of spliced RNAs harboring premature termination codons (PTC), and inhibition has been proposed as a potential therapeutic method. Conversely, NMD plays regulatory roles throughout the eukaryotic kingdom, including when protein translation is inhibited acutely as part of the integrated stress response. To define tools for endothelial evaluations of therapeutic NMD inhibition, and quantification of subtle cellular stress states, natural endothelial-expressed targets were examined via whole transcriptome RNA sequencing of primary human microvascular endothelial cells (HMECs) treated for 1h with cycloheximide, a protein translation and NMD inhibitor. Genes differentially expressed after 1h cycloheximide overlapped with genes differentially expressed many days after NMD-specific knockdown in other cell types. For endothelial cells, customized novel scripts used 255,500 exons in media-treated HMEC and 261,725 exons in cycloheximide-treated HMEC to predict 1h cycloheximide-stabilized exons. RT-PCR and RNASeq validations in other endothelial cells highlighted exon 3B of the iron transporter SLC11A2 (also known as NRAMP2/DMT1) as a novel exon in a transcript most consistently stabilized. Exact junctional alignments to SLC11A2 exon 3B were confirmed in blood outgrowth endothelial cells (BOECs) from 3 donors at mean 5.9% (standard deviation 2.0%) of adjacent constitutive exon expression, increasing 3.7-fold following 1h treatment with cycloheximide. Relevance beyond endothelial cells is supported by SLC11A2s wide expression profiles, genome-wide associations with microcytic anemia, biomarker status for poor prognosis ovarian cancer, and exon 3B sequence in RefSeq non-coding transcript NR_183176.1. The studies contribute understanding to functions affected acutely by NMD/translation inhibition and provide a stimulus for further studies in experimental, stress, and therapeutic settings.

molecular biology↗

bioRxiv2021

Heterozygous transcriptional and nonsense decay signatures in blood outgrowth endothelial cells from patients with hereditary haemorrhagic telangiectasia

Frameshift and nonsense DNA variants represent the commonest causes of monogenic inherited diseases. They usually generate premature termination codon (PTC)-containing RNA transcripts that produce truncated proteins in recombinant systems, but endogenously are subject to nonsense mediated decay. To examine native consequences of these variants, we derived cells from pre-genotyped patients. Blood outgrowth endothelial cells (BOECs) were established from individuals with hereditary hemorrhagic telangiectasia (HHT) due to a heterozygous nonsense variant in ACVRL1, ENG or SMAD4 that each encode an endothelial cell-expressed protein mediating bone morphogenetic protein (BMP)/ transforming growth factor (TGF)-{beta} signalling. RNA sequencing alignments to PTC alleles varied from 8-23% of expected, and differed between same-donor replicates. Differential gene expression analyses were validated by single cell qRT-PCR, and identification of changes in wider, disease-specific BMP/TGF-{beta} pathway components. However, the most differentially expressed genes clustered to disease-independent terms for post translational protein modification (isopeptide bond; ubiquitin-like conjugation). They were the only terms meeting Benjamini significance after clustering Bonferroni-ranked, differentially expressed genes from the 5,013 meeting 10% intraassay coefficients of variation, and significance was robust to normalisation methods. Optimised pulse chase experiments supported perturbed wildtype protein maturation, but no PTC-truncated protein was identified. Unexpectedly, BOEC cultures with highest PTC persistence were discriminated in unsupervised hierarchical clustering of low GINI coefficient invariant housekeeper genes, and patterns were compatible with higher cellular stress. The findings support a model whereby PTCs are more of a burden in stressed cells, and lead us to conclude that overlooked and varying PTC burdens contribute to biological variability.

cell biology↗

bioRxiv2021

Pre-diabetes linked microRNA miR-193b-3p targets PPARGC1A and increases lipid accumulation in hepatocytes: potential biomarker of fatty liver disease

Distinct plasma microRNA profiles associate with different disease features. Elevated plasma microRNA hsa-miR-193b-3p has been reported in patients with pre-diabetes where early liver dysmetabolism plays a crucial role. MicroRNA target databases revealed that hsa-miR-193b-3p could potentially target PPARGC1A, a master switch transcriptional co-activator that orchestrates the expression of genes involved in multiple metabolic pathways. In this study we evaluated the effects of hsa-miR-193b-3p in the human HepG2 hepatocyte cell line. We show that hsa-miR-193b-3p targets PPARGC1A/PGC1 mRNA 3UTR and reduces its expression. Overexpression of hsa-miR-193b-3p under hyperglycemia resulted in increased accumulation of intracellular lipid droplets; and quantitative real-time RT-PCR of a panel of 24 genes revealed significant disruption in the expression of genes coordinating glucose uptake, glycolysis, pyruvate metabolism, fatty acid synthesis, fatty acid oxidation, triglyceride synthesis, VLDL secretion, insulin signalling, and mitochondrial biogenesis, dynamics and function. These results provide in vitro evidence that microRNA hsa-miR-193b-3p downregulates PPARGC1A/PGC1, disrupts downstream metabolic pathways and leads to the accumulation of intracellular lipids in hepatocytes. We propose that microRNA hsa-miR-193b-3p may have potential as a clinically relevant plasma biomarker for metabolic associated fatty liver disease in a pre-diabetic dysglycemic context.

biochemistry↗