Biology subjects

Loughran, G.

Publications and source records attributed to Loughran, G..

11 recordsLinked to original sources

bioRxiv2025

Molecular architecture and diversity of StopGo/2A translational recoding

Viral 2A sequences trigger a co-translational peptide bond formation "skipping" event, termed "StopGo", to generate two separate proteins from a single open reading frame without classical termination. To investigate the mechanism of StopGo, we determined the cryo-EM structure of a mammalian ribosome positioned at the foot-and-mouth disease virus 2A (F2A) site. The structure shows how interactions between the F2A nascent chain (NC) and the ribosomal exit tunnel induce a conformational change in the peptidyl transferase center that precludes further translation elongation but instead pre-exposes the P-tRNA:F2A-NC ester bond for hydrolysis and NC release. Additionally, we bioinformatically characterized variation and host association across nearly 10,000 StopGo sequences identified in virus genomes. We expanded the canonical core motif to (D/G/C/N)(V/I)ExNPGP and identified additional rare but functional variants. We also revealed several distinct upstream motifs that we showed biochemically to be important for StopGo activity. Interestingly, although StopGo is known to be functionally active in plants, we found no evidence for natural utilization of StopGo by plant viruses. Overall, these findings provide valuable insights into a unique translation recoding mechanism, and lay foundations for further optimization of multi-gene expression in biotechnology.

molecular biology↗

bioRxiv2025

The earliest changes in the translatome upon human T cell activation

Stimulation of resting T cells triggers a rapid transition into an activated state, characterized by significant changes in phenotype, metabolism and secretory function. To explore the earliest responses, we examined transcriptional and translational changes in isolated human T cells within the first four hours post-stimulation. Polyclonal stimulation initially upregulates cytokine genes while downregulating certain transcription factors and regulators. Subsequently, selective mRNA translation occurs on 5-terminal oligopyrimidine (TOP) motif-containing mRNAs and ATF4, alongside global transcriptional reprogramming involving alternative transcription and splicing. Notably, both stimulated and unstimulated T cells undergo dynamic gene expression changes over time, reflecting adaptation to in vitro conditions and the loss of in vivo homeostatic signals. This includes heightened translation initiation stringency, particularly in non-stimulated cells. Thus, stress responses induced by standard T cell isolation protocols must be considered when investigating T cell signaling and activation.

immunology↗

bioRxiv2025

Comprehensive analysis of yeast +1 ribosomal frameshifting unveils a novel stimulator affirming two distinct frameshifting mechanisms

Ribosomal frameshifting is an important, albeit rare, mRNA decoding mechanism that generally allows the synthesis of a single protein from two different reading frames. For most +1 frameshifting cases, the mechanism is commonly presumed to involve dissociation of the P-site tRNA from its cognate codon followed by its movement to the +1 codon, setting the new +1 frame for incoming tRNAs. This movement is stabilized by P-site tRNA pairing with the +1 codon. However, in several occurrences in the yeast Saccharomyces cerevisiae, P-site tRNA re-pairing with the +1 codon is impossible. Two alternative hypotheses exist explaining this observation. One model suggests that +1 frameshifting occurs according to a common mechanism involving P-site movement, while its re-pairing with +1 codon is not essential. The alternative model suggests a distinct mechanism in which the A-site tRNA acceptance at the +1 codon occurs in the absence of P-site tRNA movement relative to mRNA. Here we set out to perform a comprehensive comparative analysis of all known +1 ribosomal frameshifting sites in S. cerevisiae. This included a novel case of +1 ribosomal frameshifting that we discovered during this study. It is required for the expression of LLP1 gene encoding dolichol-linked oligosaccharide pyrophosphatase. During the analysis of all frameshifting contexts, we identified a conserved RNA secondary structure located almost immediately upstream of the ABP140 frameshifting site. This structure substantially increases +1 frameshifting efficiency. The RNA stimulators location suggests that mRNA exiting the ribosome forms this structure, creating an mRNA pulling effect, thus favouring positioning of the +1 codon in the P-site. Placing the stimulator upstream of various known frameshifting sites, revealed that its stimulatory action is selective to those frameshifting sites where P-site tRNA re-pairing is possible, reinforcing the idea of two distinct mechanisms of ribosomal frameshifting.

molecular biology↗

bioRxiv2024

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active mediator of kinesin-1-dependent lysosome transport

Programmed ribosomal frameshifting is a process where a proportion of ribosomes change their reading frame on an mRNA1, rephasing the ribosome relative to the mRNA. While frameshifting is commonly employed by viruses2, very few phylogenetically conserved examples are known in nuclear encoded genes and some of the evidence is controversial3,4. Here we report a +1 frameshifting event during decoding of the human gene PLEKHM25. This frameshifting occurs at the sequence UCC_UUU_CGG, which is conserved in vertebrates and is similar to an influenza virus sequence that frameshifts with similar efficiency6,7. The new C-terminal domain generated by this frameshift forms an -helix, which relieves PLEKHM2 from autoinhibition and allows it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Reintroducing both the canonically-translated and frameshifted protein are necessary to restore normal contractile function of PLEKHM2-knockout cardiomyocytes, demonstrating the necessity of frameshifting for normal cardiac activity.

molecular biology↗

bioRxiv2023

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarity of the two states. Notably though, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM induces a senescence-like phenotype rapidly and synchronously thereby allowing the study of its core regulatory program without any confounding heterogeneity.

genomics↗

bioRxiv2022

The translating bacterial ribosome at 1.55 A resolution by open access cryo-EM

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome1-4. This complex macromolecular machine is the target of important antimicrobial drugs5, an integral line of defence against infectious diseases. Here, we describe how open access to state-of-the-art cryogenic electron microscopy facilities combined with bespoke user support offered by the newly established EMBL Imaging Centre enabled structural determination of the translating ribosome from Escherichia coli at 1.55 [A] resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enables interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 [A] resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

molecular biology↗

bioRxiv2020

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

molecular biology↗

bioRxiv2020

Tissue-specific dynamic codon redefinition in Drosophila

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Unprecedentedly efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

While near cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here we describe a rare case of highly efficient near cognate initiation. A CUG triplet located in the 5 leader of POLG mRNA initiates almost as efficiently (~60-70%) as an AUG in optimal context. This CUG directs translation of a conserved 260 triplet-long overlapping ORF, which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5 of this CUG governs the ratio between DNA polymerase and POLGARF produced from a single POLG mRNA. Functional investigation of POLGARF points to extracellular signalling. While unprocessed POLGARF resides in the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid secretion of POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ~160 million years ago due to an MIR transposition into the 5 leader sequence of the mammalian POLG gene which became fixed in placental mammals. The discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution. Significance StatementIn this study, we describe previously unknown mechanism of de novo protein-coding gene evolution. We show that the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase, is in fact a dual coding gene. Ribosome profiling, phylogenetic conservation, and reporter construct analyses all demonstrate that POLG mRNA possesses a conserved CUG codon which serves as a start of translation for an exceptionally long overlapping open reading frame (260 codons in human) present in all placental mammals. We called the protein encoded in this alternative reading frame POLGARF. We provide evidence that the evolution of POLGARF was incepted upon insertion of an MIR transposable element of the SINE family.

molecular biology↗

bioRxiv2019

Contesting the evidence for -1 frameshifting in immune-functioning C-C chemokine receptor 5 (CCR5) - the HIV-1 co-receptor

During the decoding of a subset of mRNAs a proportion of ribosomes productively shift to the -1 reading frame at a specific, slippage-prone site1. While the great majority of occurrences of the \"Programmed -1 Ribosomal Frameshifting\" (-1 PRF) involve viruses2, other mobile elements or retroelements1, a dramatic instance of utilized human cellular -1 frameshifting in a non-retroelement derived mRNA has been reported. The mRNA for which -1 frameshifting was claimed due to a stimulatory pseudoknot is that which encodes immune functioning C-C chemokine receptor 5 (CCR5), the HIV-1 co-receptor3. The publication ascribed the importance of the reporter CCR5 mRNA frameshifting directing translating ribosomes to a premature termination codon leading to mRNA destabilization via the nonsense-mediated decay pathway (NMD), rather than creating a C- ...

molecular biology↗

bioRxiv2017

TASEP modelling provides a parsimonious explanation for the ability of a single uORF to upregulate downstream ORF translation during the Integrated Stress Response

Translation initiation is the rate limiting step of protein synthesis that is downregulated during Integrated Stress Response (ISR). In our previous work (Andreev, OConnor et al 2015), we demonstrated that most human mRNAs resistant to this inhibition possess translated uORFs and in some cases a single uORF is sufficient for the resistance. Here we developed a computational model of Initiation Complexes Interference with Elongating Ribosomes (ICIER) to gain insight into the mechanism. We explored the relationship between the flux of scanning ribosomes upstream and downstream of a single uORF depending on uORF features. Paradoxically our analysis predicts that reducing ribosome flux upstream of certain uORFs increases initiation downstream. The model reveals derepression of downstream translation as general mechanism of uORF-mediated stress resistance. It predicts that stress resistance can be achieved with long or slowly translated uORFs that do not favor high levels of translation re-initiation and start with non-leaky initiators.

bioinformatics↗