Biology subjects

Neil, C. R.

Publications and source records attributed to Neil, C. R..

4 recordsLinked to original sources

bioRxiv2024

One-Size-Fits-Many: Antisense oligonucleotides for rescuing splicing mutations in hotspot exons

Mutations that impact splicing play a significant role in disease etiology but are not fully understood. To characterize the impact of exonic variants on splicing in 71 clinically-actionable disease genes in asymptomatic people, we analyzed 32,112 exonic mutations from ClinVar and Geisinger MyCode using a minigene reporter assay. We identify 1,733 splice-disrupting mutations, of which the most extreme 1-2% of variants are likely to be deleterious. We report that these variants are not distributed evenly across exons but are mostly concentrated in the [~]8% of exons that are most susceptible to splicing mutations (i.e. hotspot exons). We demonstrate that splicing defects in these exons can be reverted by ASOs targeting the splice sites of either their upstream or downstream flanking exons. This finding supports the feasibility of developing single therapeutic ASOs that could revert all splice-altering variants localized to a particular exon.

molecular biology↗

bioRxiv2022

Large scale functional screen identifies genetic variants with splicing effects in modern and archaic humans

Humans co-existed and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,224 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 969 exonic splicing mutations (ESMs) that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than in Neanderthals. Adaptively introgressed variants were enriched for moderate effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a novel tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a novel Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide novel insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.

genomics↗

bioRxiv2021

Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

Platelets, small hemostatic blood cells, are derived from megakaryocytes, although the generation process is not clear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates very large numbers of platelets, up to 3,000 per megakaryocyte. Despite their large size, megakaryocytes were able repeatedly to passage through the lung vasculature, leading to enucleation and fragmentation to generate platelets intravascularly. Using the ex vivo lung and a novel in vitro microfluidic chamber we determined the contributions of oxygenation, ventilation and endothelial cell health to platelet generation, and showed a critical role for the actin regulator TPM4. One-Sentence SummaryMegakaryocytes form platelets intravascularly in the lung, dependent upon oxygenation, endothelium and megakaryocyte TPM4

physiology↗

bioRxiv2020

L-bodies are novel RNA-protein condensates driving RNA transport in Xenopus oocytes

RNP granules are membrane-less compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are transported as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here, we demonstrate that vegetal transport RNPs represent a new class of cytoplasmic RNP granule, termed Localization-bodies (L-bodies). We show that L-bodies are multiphase RNP granules, containing a dynamic protein-containing phase surrounding a non-dynamic RNA-containing substructure. Our results support a role for RNA as a critical scaffold component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

cell biology↗