FEBS Letters

The synthesis of mitochondrial-encoded polypeptides is an essential process, primarily regulated at the posttranscriptional level. In yeast, many regulatory factors have been described as acting in proximity to the mitoribosome to promote efficient translation; however, the precise mechanisms by which these components function remain largely unknown. Here, we expand on findings concerning a previously studied mitoribosome interactor, Mrx9, which is found in large expressosome-like assemblies of mitoribosome clusters. Mrx9 was initially linked to mitochondrial translation and was suggested to be associated with the splicing of COX1 and COB transcripts. Our current data show that Mrx9 is associated with the PHB/m-AAA complex at the polypeptide exit tunnel of the mitoribosome. Overexpression of Mrx9 impairs the proteolytic functions of Yta10 and Yta12 within the prohibitin complex, leading to splicing defects; accumulation of aberrant polypeptides; and a noticeable impairment in the processing of the essential mitoribosomal protein bL32m. These findings support a regulatory role for Mrx9 in the PHB/m-AAA complex by modulating the activities of both Yta10 and Yta12.

Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.

Life evolved under broad-spectrum sunlight (300-3500 nm), but most modern light-emitting diodes (LEDs) emit within a restricted visible range (350-650 nm) leaving lighting environments that are short-wavelength dominant. We ask if chronic 450nm exposure dominant in LEDs alters metabolism and organ integrity in ageing mice environmentally exposed at 13 mW/cm{superscript 2} for 5 h/day over 40 weeks, at an intensity within the range of human environmental exposures. Mice exhibited progressive weight gain despite reduced food intake, with altered glucose tolerance and insulin sensitivity over time. White adipose tissue mass increased, while brown adipose tissue weight and UCP1 protein levels remained unchanged. Relative organ weights (heart, liver, kidney, testis) were each reduced by ~10%, with histopathology revealing fat deposition, hepatocellular degeneration, renal tubular damage, steatosis, and impaired testicular architecture. These findings demonstrate that chronic 450 nm light exposure is associated with metabolic alterations and multiorgan pathology in mice.

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.

N2-methylguanosine (m2G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from Thermococcus kodakarensis contains m2G at position 67. We previously proposed that the tRNA m2G methyltransferase Trm14 is responsible for m2G67 formation in tRNATrp from T. kodakarensis, although Trm14 was originally identified as the enzyme catalyzing m2G6 formation in tRNACys in Methanocaldococcus jannaschii. Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant T. kodakarensis Trm14 revealed that the enzyme catalyzes m2G formation at positions 6 and 67 in T. kodakarensis tRNACys and tRNATrp transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m2G6 and m2G67 in native tRNACys and tRNATrp, respectively, from a T. kodakarensis trm14 gene disruptant strain, providing direct evidence for the dual-site specificity of T. kodakarensis Trm14. The growth phenotype of the trm14 gene disruptant strain was comparable to that of the wild-type strain. In contrast, a trm14/trm11 double disruptant, in which trm11 encodes the tRNA m2G10/m22G10 methyltransferase, exhibited severe growth retardation at 95 {degrees}C. This suggests that m2G6/m2G67 and m2G10/m22G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 T. kodakarensis tRNA transcripts. Furthermore, we found that recombinant M. jannaschii Trm14 methylated both positions. In contrast, the bacterial ortholog TrmN modified only position 6 in tRNA. Overall, this study expands our understanding of archaeal Trm14 by demonstrating its broader substrate specificity and the physiological significance of these modifications under hyperthermophilic conditions.

Ferritins are ubiquitous iron homeostasis proteins found across the tree of life that form conserved 24-subunit cages with octahedral (4-3-2) symmetry. New types of ferritins and ferritin-like proteins are being continuously discovered, such as mini-bacterioferritins, which form smaller shells of 12 subunits, and double-ferritin-fold proteins, which act as ferroxidases but do not form shells. Here, we describe double-ferritin-fold proteins from Asgard archaea, dubbed dFTNs, and determine Cryo-EM structure of a representative from Candidatus Heimdallarchaeum endolithica. The protein forms a dodecameric shell with tetrahedral (2-3) symmetry. N-terminal (NTD) and C-terminal (CTD) domains are bridged by an ordered linker and are related by two-fold rotational pseudosymmetry. C-terminal -helix (helix E) that forms the four-fold channel in classic ferritins is repositioned to be the helix 2 out of 5 ferritin domain -helices in dFTN, with two such helices from NTD and two helices from CTD forming a pseudo-four-fold symmetry structural element. Four three-fold channels are formed by NTDs, and four other such channels are formed by CTDs. The overall arrangement of dFTN ferritin domains is similar to that of protomers in classic ferritin shells. Altogether, our findings expand the range of known ferritin family proteins and provide insight into Asgard archaea iron metabolism.

The thermal stability of collagen triple helices is strongly influenced by the amino acid sequence of the repeating Gly-X-Y tripeptides, yet how these residue-specific interactions are integrated within an extended triple helix to determine thermal behavior remains poorly understood. Here, we addressed this question using recombinant collagen mimetic peptides (rCMPs) containing extended native sequences from the 1(I) and 2(I) chains of human type I collagen. Triple-helix formation was nucleated by a C-terminal foldon domain and further stabilized by interchain disulfide crosslinking, allowing the apparent melting temperature (T) to reflect interactions within the triple-helical domain independent of nucleation. The stabilizing effects of Pro and Y-position Arg identified in host-guest peptides were largely preserved in extended triple helices, whereas the proposed Lys-Gly-Glu (KGE) interchain salt bridge produced little measurable stabilization, demonstrating the influence of sequence context. Remarkably, identical triple-helical sequences exhibited markedly different thermal behavior when unfolding was initiated under different conditions. Nevertheless, extended triple helices differing substantially in sequence and length retained an apparently two-state thermal transition. These findings support a mechanism in which unfolding is preferentially initiated within regions of lower intrinsic stability, while the continuity of the triple helix couples neighboring regions into a cooperative unfolding process throughout the helix. This mechanism provides a plausible explanation for the longstanding paradox that extended collagen triple helices exhibit persistent sequence-dependent thermodynamic heterogeneity despite a two-state thermal transition, and a framework for investigating how sequence-dependent stability contributes to the structure and function of collagen molecules. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/741348v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f311aorg.highwire.dtl.DTLVardef@160dc76org.highwire.dtl.DTLVardef@29e989org.highwire.dtl.DTLVardef@1a3279c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of alpha-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

BackgroundTuberous Sclerosis Complex (TSC) is a genetic disorder caused by variants in TSC1 or TSC2, leading to mTORC1 hyperactivation and autophagy suppression. Although TSC tumorigenesis typically follows a "two-hit" model, the role of TSC2 haploinsufficiency in autophagy regulation remains unclear. We evaluated autophagy markers in haploinsufficient and gene-edited TSC2 primary cells and investigated the role of metformin in modulating autophagy levels. MethodsPrimary fibroblast cultures were obtained from one healthy individual and three from patients carrying heterozygous germline TSC2 variants: the pathogenic variants c.1008T>G and c.4375C>T.A variant of uncertain significance (VUS) c.724A>T. CRISPR/Cas9-RNP editing was used to model loss of heterozygosity (LOH) in cell pools carrying each variant. Cultures were treated with rapamycin, HBSS, metformin, bafilomycin A1, or vehicle controls, and autophagy was assessed by autolysosomes formation by flow cytometry (acridine orange) and autophagosomes immunofluorescence (LC3 and p-S6K). ResultsIn wild-type cells, only HBSS increased autophagy-positive (acridine orange-positive) cells versus control (15.6% vs. 7.5%; p=0.003). In heterozygous pathogenic cells, rapamycin and metformin increased autophagic cells: c.1008T>G (16.2%, p=0.006; 17.6%, p=0.002) and c.4375C>T (12.5%, p=0.003; 13.3%, p=0.001), versus DMSO controls (9.2% and 7.1%, respectively). VUS c.724A>T cells, with rapamycin increasing autophagic cells (9.74% vs. 6.5%; p=0.0152). In CRISPR-edited cells, all treatments increased the number of autophagic cells compared to the heterozygous cells: c.1008T>G (rapamycin 27.1% vs. 16.7%, p<0.001; metformin 27.2% vs. 17.6%, p<0.001) and c.4375C>T (rapamycin 21.3% vs. 13.1%, p=0.0021; metformin 21.5% vs. 13.6%, p=0.0029). Editing also restored metformin responsiveness in VUS cells (12.5% vs. 8.4%; p=0.0055). Immunochemistry confirmed increased total LC3II and decreased p-S6K across treated cells compared to the control (DMSO). ConclusionThese findings demonstrate that TSC2 haploinsufficiency functionally impairs autophagy prior to second-hit loss. Metformin effectively restores autophagy with phenotypical changes of mTORC1 blockade, highlighting an accessible translational strategy to restore and induce autophagy in TSC cells.

A fraction of patients treated with radiotherapy are known to be more sensitive to ionizing radiations. Skin fibroblasts from such radiosensitive individuals exhibit a higher cellular toxicity after irradiation and a delay in DNA repair. Deciphering the molecular mechanisms underlying these cellular defects is thus of major importance. We previously observed that the transcription factor NFATc2 is expressed at a reduced level in fibroblasts from radiosensitive patients. The present work aimed to elucidate the role of NFATc2 in the regulation of DNA repair, particularly the repair of radiation-induced double-strand breaks. We demonstrate an interaction of NFATc2 with the NHEJ repair protein Ku80 and observe that the NFATc2 RHD domain is necessary and sufficient for this interaction. Moreover, we show that NFATc2-Ku80 complexes are not colocalized to DNA double-strand breaks sites suggesting an involvement upstream of the DNA repair pathway. The silencing of NFATc2 impairs the NHEJ repair activities by delaying Ku70-Ku80 interaction in the early steps of this pathway. Finally, stable over-expression of NFATc2 in patients fibroblasts partially rescues their defective DNA repair phenotype, especially in the most radiosensitive cells. Altogether, our data reveal that NFATc2 is a regulator of DNA repair in skin fibroblasts and therefore a potential modulator of cellular radiosensitivity.

Surface layers (S-layers) are paracrystalline protein lattices that form the outermost layer of the cell envelope in most archaea, providing structural support, protecting against external insults, and co-ordinating interactions with their environment. Despite their widespread occurrence, the molecular and structural details of S-layer architecture in hyperthermophilic archaea remain largely unknown. Here, we report the structure and cellular architecture of the S-layer from the hyperthermophilic archaeon Pyrobaculum arsenaticum by combining in situ electron cryotomography with single-particle electron cryomicroscopy, AlphaFold modelling, and peptide-fingerprinting mass spectrometry. We show that the S-layer is formed by an uncharacterised 292-kDa S-layer protein (SLP) extending 37 nm from the cytoplasmic membrane, making it, to our knowledge, the largest SLP structurally characterised to date. This SLP has a remarkable multidomain architecture comprising 19 immunoglobulin-like domains, 14 canonical and five non-canonical, organised into a lattice-forming core, a stalk, and a unique crown domain that stabilise the S-layer. Comparative genomic analyses unearthed homologous colossal SLP candidates across Thermoproteota, indicating that this distinctive architecture is conserved across diverse archaeal lineages. Together, our findings provide a structural framework for understanding the cell-surface organisation in hyperthermophilic archaea and suggest that these colossal S-layers represent a specialised adaptation to life at high temperatures.

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

The tRNAs are non-coding RNAs (ncRNAs) known for their classical role in decoding mRNAs. Analysis of tRNA sequences from Escherichia coli revealed pairwise similarities for different combinations of tRNAs, as well as complementarities between some tRNA pairs. To explore the physiological significance of the complementarities between the tRNAs, we investigated the pair of tRNAs encoded by lysT (encoding an abundant tRNA, tRNALysT), and argU (encoding a rare tRNA, tRNAArgU). We show that tRNALysT and tRNAArgU anneal to form a heterodimer in vitro. The heterodimerisation is prevented by the presence of DNA oligomers complementary to the interacting sequences, in a dose dependent manner. Consistent with the notion of sequestration of tRNAArgU by tRNALysT, while the overexpression of tRNAArgU did not impact the culture growth, that of tRNALysT did. The tRNALysT mediated inhibition of the culture growth was enhanced at a lower temperature. The AGA minigene (decoded by tRNAArgU) mediated toxicity and hybrid phage ({lambda}imm-P22) growth on ssrA (tmRNA) strains was also consistent with the sequestration of tRNAArgU by tRNALysT. Also, we observed tRNA-derived fragments (tRFs) from tRNALysT and tRNAArgU, which too might facilitate tRNAArgU sequestration. Taken together, these observations support a novel regulatory role of the evolutionary conserved complementarities between tRNAs.

X-linked amyotrophic lateral sclerosis (ALS) implicated Ubiquilin 2 (UBQLN2) is expressed from a single exon. Here, we show that human UBQLN2 mRNA is alternatively spliced by virtue of a cryptic exonic intron (exitron) spanning the 5' untranslated region and the 5' end of UBQLN2 coding sequence. Splice-out of this exitron generates a spliced UBQLN2 (UBQLN2-Sp) transcript that is translated from codon M243 to produce an N-terminally truncated UBQLN2 isoform (UBQLN2-M243) with reduced stability, diminished proteasome targeting, and altered aggregation behavior following the introduction of ALS mutations. The RNA-binding proteins SRSF1 and PTBP1 control UBQLN2 splicing through binding to splice donor-proximal motifs, while TDP-43 was implicated as an indirect splicing repressor. UBQLN2 splicing was elevated and inversely correlated with UBQLN2 gene expression in the medial motor cortex of male ALS patients. These findings suggest that alternative splicing regulates UBQLN2 gene dosage and function, which may impact UBQLN2-ALS proteinopathy.

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

Hematopoietic stem cells (HSCs) give rise to all blood cell lineages and possess long-term self-renewal potential. HSCs undergo symmetric division for their expansion and asymmetric division to generate one HSCs and one progenitor cells which contribute to production of mature blood cells. The midbody is a structure which is formed in the center of the intercellular bridge during cytokinesis. However, the midbody is either asymmetrically inherited by one daughter cell or symmetrically released after cell division, whether these distinct patterns of midbody inheritance influence HSC fate remain poorly understood. In this study, we designed a fusion protein hmKO2 and MgcRacGAP which is a component of midbody. We then traced the midbody inheritance during cell division and the future cell fates of HSC daughters after division by time-lapse imaging. As a result, we found that the midbody release correlated with the delay of the time to the next division but not to the lineage potential of HSCs, indicating the possibility that midbody remnant plays some roles in cell cycle progression. HighlightHematopoietic stem cells exhibit a low frequency of midbody inheritance. Midbody inheritance does not affect the lineage potential of daughter cells. Midbody loss is associated with delayed entry into the next cell cycle.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Magnetic fields can modulate the function of certain flavoproteins through the radical pair mechanism, in which they influence the spin evolution of a coherent pair of radicals. This process can result in magneto-fluorescence, in which magnetic fields modulate the intensity of fluorescence emitted from the flavin cofactor. The prevalence of this property across protein families and domains of life, however, remains poorly characterized. In canonical Light-Oxygen-Voltage (LOV) domains, a conserved cysteine forms an adduct with the flavin that leads to downstream signaling. Mutating this cysteine instead yields signaling through the neutral semiquinone radical form, and the same mutation was crucial for enhancing magneto-fluorescence in the engineered protein MagLOV2. We therefore hypothesized that natural LOV domains that lack this conserved cysteine may exhibit magneto-fluorescence. Using a custom magneto-fluorescence imaging platform, we measured the fluorescence of E. coli colonies expressing three such domains, as well as a single mutant of one of them, under a switched external field. HsuLOV, from a halophilic archaeon, exhibited magneto-fluorescence, as did a single mutant of BAT-LOV, from a second halophilic archaeon. The domain amb2291 from magnetotactic bacteria, on the other hand, showed no detectable response under our illumination conditions. Magneto-fluorescence is therefore not exclusive to engineered proteins but is present in natural cysteine-less LOV domain sequences. The proteins reported here are, to our knowledge, the first magnetosensitive proteins reported from archaea. This suggests that radical-pair magnetosensitivity may be more widespread across LOV domains than previously appreciated.