Biology subjects

Kearns, L.

Publications and source records attributed to Kearns, L..

4 recordsLinked to original sources

bioRxiv2024

Simultaneous mapping of RNA secondary, tertiary, and quaternary structure in living cells by multi-site DMS probing

Identifying tertiary structures and protein binding sites on RNA molecules remains a key challenge in RNA biology. We report a new chemical probing strategy termed multi-site DMS-MaP (msDMS-MaP) that exploits base tautomerization during mutational profiling reverse-transcription to measure typically invisible DMS-induced modifications at the N7 position of G. These N7-G modifications are now resolved concurrently with conventional N1 and N3 modifications with only minor modifications to established protocols, providing a multi-dimensional, single-molecule readout of RNA structure. We show that N7-G reactivity specifically reports on RNA tertiary and quaternary structure, enabling identification of diverse, functionally significant motifs such as cooperatively folding tertiary domains and protein binding sites in living cells. We apply msDMS-MaP to obtain a map of the quaternary structural ensemble of the 7SK non-coding snRNP, revealing unique protein binding sites across three 7SK structural isoforms. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.

biochemistry↗

bioRxiv2021

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

neuroscience↗

bioRxiv2021

Retinal ganglion cell-specific genetic regulation in primary open angle glaucoma

To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared to those from healthy individuals. We performed single-cell RNA-sequencing of a total of 330,569 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 2,235 significant loci across all cell types, 58 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 54 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.

genetics↗

bioRxiv2018

Congenital glaucoma with anterior segment dysgenesis in individuals with biallelic CPAMD8 variants

PurposeCongenital glaucoma is a significant cause of irreversible blindness. In some instances glaucoma is associated with developmental abnormalities of the ocular anterior segment, which can impair drainage of aqueous humor, leading to an increase in intraocular pressure.\n\nMethodsGenome sequencing was performed on a parent-proband congenital glaucoma trio, with exome sequencing of 79 additional individuals with suspected primary congenital glaucoma.\n\nResultsWe describe a unique ocular anterior segment dysgenesis associated with congenital glaucoma in four individuals from three unrelated families. In each case, disease was associated with compound heterozygous variants in CPAMD8, a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation of key drainage structures and the development of high intraocular pressure and glaucoma.\n\nConclusionsThis study reveals a unique genetic cause of childhood glaucoma, and expands the phenotypic spectrum of CPAMD8-associated ocular disease.

genetics↗