Biology subjects

Digby, H.

Publications and source records attributed to Digby, H..

4 recordsLinked to original sources

bioRxiv2026

N6-methyladenosine (m6A) RNA modifications control the antiviral response to influenza A virus infection by limiting IFITM2/3 expression

N6-methyladenosine (m6A) RNA modifications regulate diverse cellular processes, including the response to virus infection. However, how m6A controls antiviral gene expression and whether effects on viral replication are due to direct modification of viral RNA or regulation of cellular transcripts remain unclear. Herein, we analysed how m6A modifications control the cell-autonomous innate immune response to influenza A virus (IAV) infection. Depletion or inhibition of the m6A writer METTL3 or the m6A reader YTHDF2 restricts IAV replication. However, using a quantitative method to identify m6A sites, we did not detect substantial m6A modification of viral RNA. In contrast, many IAV-induced cellular transcripts were m6A modified in their coding region, including those that encode pattern recognition receptors and direct antiviral proteins. Inhibition of interferon signalling restored IAV replication after METTL3 inhibition, highlighting that m6A controls the antiviral response to infection. METTL3 inhibition increased expression of the paralogous antiviral proteins IFITM2 and IFITM3, which were required for the antiviral activity of the METTL3 inhibitor. We propose that m6A modifications regulate the expression of antiviral proteins to limit the magnitude of their induction and balance antiviral activity with maintaining cellular homeostasis.

microbiology↗

bioRxiv2023

Creation of de novo cryptic splicing for ALS/FTD precision medicine

A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe "TDP-REG", a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF in vitro and in vivo, including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the UNC13A cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals. One-Sentence SummaryWe engineer TDP-43-regulated cryptic exons, enabling exceptionally precise activation of gene therapies in diseased neurons.

molecular biology↗

bioRxiv2023

Mutual homeostasis of charged proteins

Protein dosage is regulated to maintain cellular homeostasis and health. The dosage of proteins containing disordered low complexity domains (LCDs) must be particularly well-controlled to prevent aberrant disease, yet no mechanism to maintain homeostasis has been identified1, 2. Here we report a mutual homeostatic mechanism that controls the concentration of such proteins, termed interstasis, in which proteins with similar LCDs co-regulate their combined dosage through collective negative feedback. We focused on the mechanism that exploits the fundamental multivalency of GA-rich RNA regions that encode charged LCDs, including those with arginine-enriched mixed charge domains (R-MCDs). Modest variations in the abundance of an R-MCD protein change the properties of nuclear speckles, a protein-RNA condensate, selectively trapping multivalent GA-rich mRNAs to promote their nuclear retention. This interstasis depends on conserved codon biases, shared by amniotes, which enhance the multivalency of GA-rich regions encoding charged LCDs. The threshold of interstasis is modulated by CLK kinases, which affect the nuclear speckle localisation of proteins such as TRA2B, key binder of GA-rich RNAs. Notably, many classes of LCDs are encoded by RNA regions containing multivalency-enhancing codon biases, each preferentially bound by specific proteins, suggesting that interstasis might co-regulate many classes of functionally related LCD-containing proteins through dose-sensitivity of various types of protein-RNA condensates.

molecular biology↗

bioRxiv2023

Abundant capped RNAs are derived from mRNA cleavage at 3'UTR G-Quadruplexes

The 3 untranslated region (3UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis gene expression (CAGE), a method for the detection of capped 5 ends of mRNAs, additionally reveals a large number of apparently 5 capped RNAs derived from 3UTRs. Here we provide the first direct evidence that these 3UTR-derived RNAs are indeed capped and often more abundant than the corresponding full-length mRNAs. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA knockdowns, we find that these 3UTR-derived RNAs likely originate from AGO2-mediated cleavage, and most often occur at locations with potential to form RNA-G-quadruplexes and are enriched by RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validates several 3UTR-derived RNAs, demonstrates their abundance and shows that they tend not to co-localise with the parental mRNAs. We also find that production of 3UTR-derived RNA could explain the previously reported role of a 3UTR G-quadruplex in regulating the production of APP protein. Taken together, we provide new insights into the origin and abundance of 3UTR-derived RNAs, show the utility of CAGE-seq for their quantitative detection, and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/538568v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3f2ca7org.highwire.dtl.DTLVardef@18ca1b6org.highwire.dtl.DTLVardef@1ccdd8dorg.highwire.dtl.DTLVardef@e58938_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗