Biology subjects

Correa, I. R.

Publications and source records attributed to Correa, I. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Semirandom DNA adducts regulate a filamentous defence-associated reverse transcriptase

Retrons and several defence-associated reverse transcriptases (DRTs) synthesize non-genomic DNA for bacteriophage immunity. In some instances, this non-genomic DNA is of undefined, semirandom sequence. How undefined DNA sequences impart antiphage defence is not known. Here we report the cryo-EM structure and functional characterization of the DRT1 antiphage defence system. We show that DRT1 performs template-free, protein-primed DNA synthesis to generate semirandom DNA adducts. DNA synthesis activates the nitrilase domain of DRT1 while DNA adducts drive assembly of quiescent DRT1 filaments. Filamentous DRT1 is comprised of domain-swapped C-termini that are entwined, forming pseudoknots between tetrameric stacks. This configuration occludes the apo-nitrilase active site, resulting in a dormant state. Bacteriophage escape mutants identify a T4 single-stranded DNA helicase required for DRT1 activity. Functionally, DRT1 resembles a minimal retron where a single gene produces an RT, effector, and non-genomic antitoxin DNA.

microbiology↗

bioRxiv2025

Association Studies of Phage Metagenomes from Aquatic Environment Identify Known and Potentially New Key Players in the 2,6-Diaminopurine Biosynthesis Pathway

Advances in next-generation sequencing (NGS) and bioinformatics have expanded the potential of metagenomic studies, particularly for unculturable organisms such as phages with heavily modified genomes. In the evolutionary arms race between phages and their hosts, some phages develop a strategy to fully modify their DNAs to evade host-encoded antiphage defenses. Here, we developed a restriction-enzyme based method to selectively enrich all phages containing 2-aminoadenine (diaminopurine or dZ) directly from purified metagenomic DNA from environmental samples. Applying our previously established metagenomic genome-phenome association (metaGPA) pipeline, we identified 116 protein domains significantly associated with dZ phages. The top candidate domains fully recapitulated the phage-encoded dZ biosynthetic pathway. Study of individual enzymes, including PurZ and YfbR-like dATPase at residue resolution revealed key amino acids for the distinction between dZ and dA substrate. Additionally, we identified and validated two novel DNA polymerases with enhanced dZ incorporation activity and uncovered nucleoside 2-deoxyribosyltransferase (NDT) as a potential new component of the dZ biosynthetic pathway. Collectively, these findings refine current models of the dZ biosynthesis pathway, potentially reveal previously unrecognized enzymatic components, and expand the understanding of phage biology with implications for synthetic biology and DNA engineering.

genomics↗

bioRxiv2025

Coronavirus NSP14 Drives Internal m7G Modification to Rewire Host Splicing and Promote Viral Replication

SARS-CoV-2, the causative agent of COVID-19, manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral nonstructural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (GTP) to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14s N7-methyltransferase (N7-MTase) domain and is enhanced by interaction with NSP10. Internal m7G modification by NSP14 predominantly occurs in the nucleus and is conserved across alpha-, beta- and gamma-coronaviruses. Mechanistically, we show that this RNA modification disrupts normal splicing by promoting intron retention and generating novel splice junctions, particularly in genes regulating genome stability, RNA metabolism and nuclear processes. Importantly, inhibition of m7G modification, through pharmacological targeting of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks host transcriptomic machinery to support infection. Our findings reveal a previously unrecognized epitranscriptomic mechanism by which coronaviruses reprogram host gene expression and suggest that NSP14-induced m7G modification is a potential therapeutic target.

microbiology↗

bioRxiv2025

Multifaceted mRNA analysis using programmed RNA cleavage byMucilaginibacter paludis Argonaute

Argonaute (Ago) endonucleases are ubiquitous across all domains of life and, like Cas nucleases, can be programmed by guide oligonucleotides to target and cleave specific nucleic acid sequences. However, unlike Cas enzymes, Agos are not restricted by sequence requirements such as protospacer adjacent motifs (PAMs) enhancing their utility as molecular tools. This study demonstrates diverse applications of the DNA-guided RNA-endonuclease Mucilaginibacter paludis Argonaute (Mpa Ago) for in vitro mRNA processing, e.g., 3 end polishing, and for mRNA analysis in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS). Applications of the latter include the comprehensive assessment of 5 capping efficiency, 3 poly(A) tail length and composition, one-pot multiplexed cleavage for RNA sequencing, modification detection, and fingerprinting.

molecular biology↗

bioRxiv2023

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by a small number of well characterized enzymes. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We systematically assayed the activity of 175 putative cytosine deaminases on an unprecedented variety of substrates with epigenetically relevant base modifications. We found enzymes with high activity on double- and single-stranded DNA in various sequence contexts including novel CpG-specific deaminases, as well as enzymes without sequence preference. We also report, for the first time, enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. Using a newly discovered non-specific, modification-sensitive double-stranded DNA deaminase, we developed a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method. SEM-seq enables accurate, high-coverage, base-resolution methylome mapping of scarce biological material including clinically relevant cell-free DNA (cfDNA) and single-cell equivalent 10 pg input DNA. Using SEM-seq, we generated highly reproducible base-resolution 5mC maps, accounting for nearly 80% of CpG islands for a low input human cfDNA sample offering valuable information for identifying potential biomarkers for detection of early-stage cancer and other diseases. This streamlined protocol will enable robust, high-throughput, high-coverage epigenome profiling of challenging samples in research and clinical settings.

molecular biology↗

bioRxiv2022

GSK-3484862 targets DNMT1 for degradation in cells

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires Uhrf1, an accessory factor of Dnmt1 with E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting both coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner. HighlightsO_LIGSK-3484862 targets DNMT1 for protein degradation in a wide-range of cancer cell lines, without a decrease in DNMT1 mRNA levels C_LIO_LIDNMT1 depletion leads to a >50% loss of global DNA methylation in cells within 2-days of treatment with GSK-3484862 C_LIO_LIGSK-3484862-induced DNMT1 degradation is proteasome-dependent C_LIO_LIIn mESCs, Uhrf1 is required for GSK-3484862 to induce Dnmt1 degradation C_LI

cancer biology↗

bioRxiv2022

RNase H-based analysis of synthetic mRNA 5-prime cap incorporation

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5 cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5 cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5 end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5 cleavage products, and LC-MS intact mass analysis. In this communication, we describe 1) a framework to design site-specific RNA cleavage using RNase H; 2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5 cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

biochemistry↗

bioRxiv2022

RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely regulated by structured RNA elements prevalent in long noncoding and other cellular RNAs.

biochemistry↗

bioRxiv2021

The structural basis of mRNA recognition and binding by eukaryotic pseudouridine synthase PUS1

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or PUS enzymes include those that rely on guide RNA molecules and others that function as stand-alone enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 [A] resolution. The enzyme recognizes and binds both strands in a helical base-paired RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

biophysics↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

Multi-Factor Authentication of Potential 5' Splice Sites by the Saccharomyces cerevisiae U1 snRNP

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5 splice site (5 SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5 SS recognition have largely been done in vivo or in cellular extracts where the U1/5 SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5 SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5 SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5 SS and preferentially accumulate at these sequences rather than on close cognates. IMPACT STATEMENTThe yeast U1 snRNP recognizes multiple features of target RNAs to reversibly identify splicing-competent 5 splice sites.

biochemistry↗

bioRxiv2021

Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of glucose homeostasis and has been successfully targeted for the treatment of type 2 diabetes. Recently described biased GLP-1R agonists with selective reductions in {beta}-arrestin versus G protein coupling show improved metabolic actions in vivo. However, two prototypical G protein-favouring GLP-1R agonists, P5 and exendin-F1, are reported to show divergent effects on insulin secretion. In this study we aimed to resolve this discrepancy by performing a side-by-side characterisation of these two ligands across a variety of in vitro and in vivo assays. Exendin-F1 showed reduced acute efficacy versus P5 for several readouts, including recruitment of mini-G proteins, G protein-coupled receptor kinases (GRKs) and {beta}-arrestin-2. Maximal responses were also lower for both GLP-1R internalisation and the presence of active GLP-1R-mini-Gs complexes in early endosomes with exendin-F1 treatment. In contrast, prolonged insulin secretion in vitro and sustained anti-hyperglycaemic efficacy in mice were both greater with exendin-F1 than with P5. We conclude that the particularly low acute efficacy of exendin-F1 and associated reductions in GLP-1R downregulation appear to be more important than preservation of endosomal signalling to allow sustained insulin secretion responses. This has implications for the ongoing development of affinity- versus efficacy-driven biased GLP-1R agonists as treatments for metabolic disease.

pharmacology and toxicology↗

bioRxiv2021

A Genome-Phenome Association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA

Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Association (MetaGPA) study framework, which allows to link genetic information in metagenomes with a dedicated functional phenotype. We applied MetaGPA to identify enzymes associated with cytosine modifications in environmental samples. From the 2365 genes that met our significance criteria, we confirm known pathways for cytosine modifications and proposed novel cytosine-modifying mechanisms. Specifically, we characterized and identified a novel nucleic acid modifying enzyme, 5-hydroxymethylcytosine carbamoyltransferase, that catalyzes the formation of a previously unknown cytosine modification, 5-carbamoyloxymethylcytosine, in DNA and RNA. Our work introduces MetaGPA as a novel and versatile tool for advancing functional metagenomics.

genomics↗

bioRxiv2021

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist

ObjectiveGlucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study we investigated the cellular and metabolic effects of modulating the balance between G protein activation and {beta}-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. MethodsDipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify effects on glucose homeostasis and weight loss. ResultsLigand-specific reductions in {beta}-arrestin-2 recruitment led to reduced GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide in spite of a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and {beta}-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. ConclusionsDiminishing {beta}-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

pharmacology and toxicology↗

bioRxiv2020

Identification of high confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoform scaffolds among [~]4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nucleotide oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for ionic current signatures consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.

genomics↗

bioRxiv2020

Impact of N-terminally substituted glucagon family receptor agonists on signal bias, trafficking and downstream responses

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) are important regulators of insulin secretion and energy metabolism. Recently described GLP-1 receptor agonists showing signal bias in favour of cyclic AMP over {beta}-arrestin-2 recruitment have delivered promising results in preclinical studies. Here we first sought to establish the role of {beta}-arrestins in the control of intracellular signalling and trafficking responses at the closely related GLP-1, GIP and GCG receptors, through studies performed in cells depleted of both {beta}-arrestin isoforms. We also generated analogues of GLP-1, GCG and GIP which in some cases showed selective reduction in {beta}-arrestin-2 recruitment versus cAMP signalling compared to the parent peptide. Despite reduced acute signalling potency and/or efficacy, some biased GLP-1 and GIP analogues increased maximal sustained insulin secretion from INS-1 832/3 clonal beta cells, although only at high agonist concentrations. Biased GCG analogues did not affect maximal insulin release, or glucose output in hepatocytes.

pharmacology and toxicology↗

bioRxiv2020

The influence of peptide context on signalling and trafficking of glucagon-like peptide-1 receptor biased agonists

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated signalling, endocytosis and recycling, dependent both on the introduction of a His [->] Phe switch at position 1 and the specific mid-peptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/961524v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@cb425borg.highwire.dtl.DTLVardef@719e37org.highwire.dtl.DTLVardef@14a45b9org.highwire.dtl.DTLVardef@127afd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. However, bisulfite treatment damages DNA resulting in fragmentation, loss of DNA and biased sequencing data. To overcome this, we developed Enzymatic Methyl-seq (EM-seq), an enzymatic based approach that uses as little as 100 pg of DNA. EM-seq outperformed bisulfite converted libraries in all metrics examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts as well as increased representation of genomic features. These data indicate that EM-seq is more accurate and reliable than whole genome bisulfite sequencing (WGBS).

genomics↗