Biology subjects

Kerr, C. H.

Publications and source records attributed to Kerr, C. H..

6 recordsLinked to original sources

bioRxiv2023

RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

molecular biology↗

bioRxiv2022

Deletion of proU suppresses proQ phenotypes in Escherichia coli

The ProQ protein interacts as an RNA chaperone with diverse RNA molecules in Escherichia coli. ProQ is implicated in the bacterial osmotic stress response. When the osmotic pressure is high, cells maintain their hydration by accumulating organic solutes denoted osmolytes. Transporters ProP and ProU (which is ProVWX) mediate osmolyte accumulation by Escherichia coli. Mutations at proQ impair ProP activity by reducing ProP levels (the ProQ transport phenotype) but do not impair ProU activity or reduce the level of ProX. The proQ- bacteria are longer than proQ+ bacteria during growth in either low or high salinity medium and they grow slowly at high salinity (the ProQ growth phenotype). In addition, spherical cells with crescent-shaped, nucleic acid-rich foci appear and cells lyse (the ProQ morphological phenotypes). In this work, the proQ transport phenotype was suppressed by deletions of proU, or by an insertion of IS5 in proU, when proP was expressed from the chromosome or from the heterologous, plasmid-based PBAD promoter. A point mutation disrupting the Walker B motif of ProV inactivated ProU but did not suppress the transport phenotype. ProP activities and ProP levels varied in parallel, so proQ and proU act at the same level to regulate ProP expression. Deletion of the proU operon also suppressed the growth and morphological phenotypes. The proU locus may overlap the gene encoding a regulatory sRNA that acts with ProQ, contributing to cellular morphogenesis and osmotic stress tolerance, or the relationship between ProQ and proU may be indirect.

microbiology↗

bioRxiv2021

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that "superfolder" mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

molecular biology↗

bioRxiv2020

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes, such as limb and craniofacial defects as well as bone marrow failure. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining comprehensive mouse genetics and in vivo ribosome profiling, we observe limb patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos and uncover corresponding selective translational changes of transcripts controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are reconciled by the unexpected identification that p53 functions as a master regulator of protein synthesis through transcriptional activation of 4E-BP1. 4E-BP1, a key regulator of translation, in turn, facilitates selective changes in the translatome downstream of p53 and thereby explains, at least in part, how RP haploinsufficiency elicits specificity to gene expression. These results provide an integrative model to explain how in vivo tissue-specific phenotypes emerge from a mutation in a ribosome component.

developmental biology↗

bioRxiv2019

Dynamic rewiring of the human interactome by interferon signalling

BackgroundThe type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFN-stimulated genes (ISGs). Transcriptomic and biochemical approaches have established comprehensive catalogues of ISGs across species and cell types, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to delineate the effects of IFN signalling on the human proteome, culminating in the use of protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network.\n\nResultsWe identified >27,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer ISG protein synthesis.\n\nConclusionsOur map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing ISGs in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.

systems biology↗

bioRxiv2018

IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral pathogenicity

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

biochemistry↗