Biology subjects

Wright, B. W.

Publications and source records attributed to Wright, B. W..

5 recordsLinked to original sources

bioRxiv2026

Engineering circular RNA expression systems to minimize contaminating linear RNA byproducts

Circular RNAs (circRNAs) are generated by backsplicing of eukaryotic protein-coding transcripts and can regulate microRNAs and RNA binding proteins, or serve as translation templates. Their covalently closed structure confers resistance to exonuclease-mediated degradation, extending their half-life and supporting their development as RNA therapeutics. However, existing overexpression methods often yield substantial contaminating linear RNAs, limiting their utility. Here, we systematically benchmarked plasmid-based circRNA overexpression strategies in human cells, comparing spliceosome- and ribozyme-based mechanisms across constructs incorporating widely used flanking sequences. The ribozyme-based Tornado system produced the highest circRNA yield but introduced extraneous "molecular scars" into the mature product. By contrast, spliceosome-mediated circularization using introns from the Drosophila Laccase2 gene, which contain imperfect complementary repeats, produced scarless circRNA with substantially lower linear RNA contamination. Linear RNA was further reduced by engineering the primary transcript to terminate in a non-polyadenylated end, increasing its susceptibility to exonucleases. Building on this optimized system, we developed a dual-output platform co-expressing a linear fluorescent reporter alongside a circRNA from a single promoter (CIRCUS, circRNA and upstream linear system), enabling efficient screening of circRNA-driven cellular phenotypes, including site-specific A-to-I editing of target mRNAs. Together, this toolkit provides high-purity circRNA production suitable for mechanistic studies and circRNA-based therapeutic development.

molecular biology↗

bioRxiv2022

IbpAB small heat shock proteins are not host factors for bacteriophage {varphi}X174 replication

Bacteriophages exploit host proteins for successful infection. Small heat shock proteins are a universally conserved family of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Two small heat shock proteins, IbpA and IbpB, are a class of holding modulators or "holdases", which bind partially folded proteins and await ATP-driven folding chaperones for refolding. Bacteriophage {phi}X174 is a small, icosahedral, and non-tailed virus belonging to the Microviridae. During {phi}X174 infection of Escherichia coli C122, IbpA and IbpB were previously found to be the most highly upregulated host proteins, with expression levels comparable to {phi}X174 proteins. In this work, to understand the role of IbpA and IbpB during {phi}X174 infection, we used a hybrid approach of CRISPR interference and genomic knockouts to disrupt the ibpA and ibpB genes. We show that these two proteins do not appear to be necessary for efficient {phi}X174 replication, and moreover, their absence has no effect on {phi}X174 fecundity. ImportanceThe small heat shock proteins (sHsps) are universally conserved family of stress-induced molecular chaperones that prevent irreversible protein aggregation. In E. coli, the IbpA/B sHsps work together, and separately, to bind partially folded proteins and await ATP-driven folding chaperones for refolding. These proteins are highly upregulated during protein overexpression and bacteriophage infection, but their collective role in bacteriophage infection has not been investigated. Here, we show that the ibpA/B genes are dispensable for bacteriophage {phi}X174 infection, and are likely not essential host factors despite their abundance during diverse phage infections. Instead, this work points towards their role as cell wall integrity sensors, similar to the phage shock protein system, in addition to their canonical role as holdases of cytoplasmic protein.

microbiology↗

bioRxiv2020

Proteomic and transcriptomic analysis of Microviridae {varphi}X174 infection reveals broad up-regulation of host membrane damage and heat shock responses

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage {varphi}X174 is endemic to the human gut and has been studied for over seventy years but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome we have measured host Escherichia coli C proteomic and transcriptomic response to {varphi}X174 infection. We used mass spectrometry and RNA-seq to identify and quantify all 11 {varphi}X174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in {varphi}X174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the {varphi}X174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat-shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model phage. IMPORTANCEA major part of the healthy human gut microbiome are the Microviridae bacteriophage, exemplified by the model {varphi}X174 phage. Although much has been learned from studying {varphi}X174 over the last half century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the {varphi}X174 infection cycle, and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

microbiology↗

bioRxiv2020

Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction

ABSTRACTSequence overlap between two genes is common across all genomes, with viruses having high proportions of these gene overlaps. The biological function and fitness effects of gene overlaps are not fully understood, and their effects on gene cluster and genome-level refactoring are unknown. The bacteriophage ϕX174 genome has ∼26% of nucleotides involved in encoding more than one gene. In this study we use an engineered ϕX174 phage containing a genome with all gene overlaps removed, to show that gene overlap is critical to maintaining optimal viral fecundity. Through detailed phenotypic measurements we reveal that genome modularization in ϕX174 causes virion replication, stability, and attachment deficiencies. Quantitation of the complete phage proteome across an infection cycle reveals almost half the proteins display abnormal expression patterns. Taken together, we have for the first time comprehensively demonstrated that gene modularization severely perturbs the coordinated functioning of a bacteriophage replication cycle. This work highlights the biological importance of gene overlap in natural genomes and that reducing gene overlap disruption should be an integral part of future genome engineering projects.View Full Text

synthetic biology↗

bioRxiv2019

Measuring amber initiator tRNA orthogonality in a genomically recoded organism

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.{Delta}A.exp lacking all UAG stop codons was created, freeing this amber stop codon for other purposes. An engineered amber initiator [Formula] that activates translation at UAG codons is available, but little is known about this tRNAs orthogonality. Here, we combine for the first time the amber initiator [Formula]in C321.{Delta}A.exp and measure its cellular effects. We found that the [Formula]expression resulted in a nearly 200Yfold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosomeYassociated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for offYtarget translation initiation. In contrast to previous work, we show that UAGYinitiated proteins carry NYterminal methionine exclusively. Together, our results identify beneficial features of using the amber initiator [Formula]to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

synthetic biology↗