Biology subjects

England, W.

Publications and source records attributed to England, W..

5 recordsLinked to original sources

bioRxiv2026

Upregulation of Calbindin in Adult Inhibitory Neurons Reactivates Critical Period Plasticity in Mouse Visual Cortex

Critical periods are windows of peak learning performance where heightened synaptic plasticity enables fast, robust reorganization in juvenile brain circuits. Unlike adult plasticity which requires ongoing, persistent change in sensory experience, cortical representations can be rapidly changed during critical periods and have enduring effects. Transplantation of GABAergic inhibitory neurons has been shown to restore critical period plasticity to recipient circuits by triggering signalling changes within host inhibitory neurons. Here we transcriptionally profiled host inhibitory neurons in mouse primary visual cortex (V1) to detect gene expression changes during transplant-induced plasticity. Gene ontology enrichment analysis of differentially-expressed (DE) transcripts in host inhibitory neurons revealed synaptic plasticity- and inhibitory neuron development-related profiles. We assessed the protein expression of a top DE candidate, the calcium-binding protein Calbindin (Calb1), across transplant conditions and developmental stages. We found high Calb1 expression during the V1 critical period and during transplant-induced plasticity. To assess the functional significance of transplant-reactivated DE gene activity on visual cortical plasticity, we developed a set of AAVs to manipulate Calb1 expression specifically within inhibitory neurons. Using intrinsic signal imaging to measure ocular dominance plasticity, we found that Calb1 levels in V1 inhibitory neurons determine the extent of visual cortical plasticity. Our study provides evidence that direct induction of critical period-stage gene expression patterns in inhibitory neurons restores juvenile plasticity in targeted adult cortical circuits.

neuroscience↗

bioRxiv2020

Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling

Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Tagged RNAs were subsequently purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.

molecular biology↗

bioRxiv2020

Transcriptome-Wide Combinatorial RNA Structure Probing in Living Cells

RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptomewide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA.

biochemistry↗

bioRxiv2019

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Alternative splicing is widely acknowledged to be a crucial regulator of gene expression and is a key contributor to both normal developmental processes and disease states. While cost-effective and accurate for quantification, short-read RNA-seq lacks the ability to resolve full-length transcript isoforms despite increasingly sophisticated computational methods. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) bypass the transcript reconstruction challenges of short reads. Here we introduce TALON, the ENCODE4 pipeline for platform-independent analysis of long-read transcriptomes. We apply TALON to the GM12878 cell line and show that while both PacBio and ONT technologies perform well at full-transcript discovery and quantification, each displayed distinct technical artifacts. We further apply TALON to mouse hippocampus and cortex transcriptomes and find that 422 genes found in these regions have more reads associated with novel isoforms than with annotated ones. We demonstrate that TALON is a capable of tracking both known and novel transcript models as well as their expression levels across datasets for both simple studies and in larger projects. These properties will enable TALON users to move beyond the limitations of short-read data to perform isoform discovery and quantification in a uniform manner on existing and future long-read platforms.

genomics↗

bioRxiv2018

Exonic unpaired elements modulate pre-mRNA structure for splice site recognition

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short ([lsim]70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

biochemistry↗