Biology subjects

Martinez, A.

Publications and source records attributed to Martinez, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

The unfolded protein response in grapevine: abiotic and biotic stresses induce the expression of VvbZIP60, VvbZIP17, VvBIP3, and VvIRE1

A wide range of stresses can lead to the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER), a condition known as ER stress. To restore proteostasis, eukaryotic cells activate a signaling network called the unfolded protein response (UPR). In Vitis vinifera, two arms of the UPR have been identified: the IRE1/bZIP60 arm and the bZIP17 arm. Notably, no putative ortholog of bZIP28 has been found in V. vinifera, whereas this is the case for Brassicaceae species. We demonstrated that VvbZIP60 undergoes unconventional splicing upon treatment with dithiothreitol (DTT) and tunicamycin (TM), both classical ER stress inducers. Moreover, after testing several abiotic factors, we observed a strong transcriptional activation of UPR-related genes in response to heat and osmotic stresses, as well as copper exposure. Grapevine is also subject to a broad range of microbial challenges, including pathogens such as Plasmopara viticola and Botrytis cinerea. Both pathogens triggered UPR genes activation in grapevine leaves and berries. Interestingly, VvbZIP17 was also upregulated in green berries, a developmental stage associated with strong basal resistance to B. cinerea. Collectively, these findings suggest that the IRE1/bZIP60 and bZIP17 arms of the UPR are not only structurally conserved in grapevine but are also transcriptionally responsive to a variety of abiotic and biotic stresses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/669611v2_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@13e0b15org.highwire.dtl.DTLVardef@49967dorg.highwire.dtl.DTLVardef@1371bfborg.highwire.dtl.DTLVardef@10e09b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

DNAJC12 stabilizes activated phenylalanine hydroxylase and reduces the concentration of L-Phe needed for activation

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH4)-dependent enzyme that converts L-phenylalanine (L-Phe) to L-tyrosine. PAH dysfunction leads to the accumulation of L-Phe in the blood (hyperphenylalaninemia; HPA), which may reach neurotoxic levels, resulting in phenylketonuria (PKU). PKU is associated with pathogenic variants in PAH, most causing misfolding and instability, leading to decreased levels of PAH protein and activity. Recently, variants in the class C J-domain protein DNAJC12 have also been associated with HPA in patients, demonstrating the importance of protein homeostasis regulation for proper PAH function. DNAJC12 and PAH have previously been reported to interact, but the molecular and structural mechanisms behind complex formation have remained unclear. In this work, we show that DNAJC12 binds to PAH, but presents higher affinity for its L-Phe activated form, which resembles the conformation of unliganded tyrosine hydroxylase, a structurally and functionally-related enzyme that also binds to DNAJC12. At saturation, four monomers of DNAJC12 bind and stabilize the PAH tetramer, protecting it from aggregation and lowering the L-Phe concentration necessary for substrate-induced activation, without affecting the interaction of the enzyme with its cofactor BH4. Importantly, DNAJC12 also stabilizes and delays the aggregation of the PKU-associated variant PAH-p.R261Q. Furthermore, L-Phe activated wild-type or variant PAH is required to stimulate Hsc70 ATPase activity. SIGNIFICANCE STATEMENTDeficiencies in the cochaperone DNAJC12 have recently been linked to hyperphenylalaninemia, dystonia and intellectual disabilities as DNAJC12 regulates the proteostasis of the aromatic amino acid hydroxylases, including phenylalanine hydroxylase (PAH). This study explores the mechanisms of the PAH:DNAJC12 interaction and examines the functional effects of their complex formation on PAH activity and stability. These findings enhance our understanding on the pathogenic mechanisms behind DNAJC12 variants and provide insights that could guide the development of drugs targeting this protein-protein interaction.

biophysics↗

bioRxiv2025

Tropical montane and temperate hummingbirds incubate in small installments over long periods

Life-history strategies and development are shaped by climate and predation. We examined how temperature and nest survival drive incubation behavior in 104 nests of 34 hummingbird species across 2,939m of elevation in the Peruvian and Colombian Andes. At high elevations, cold temperatures led to more frequent, brief incubation bouts and extended incubation periods; survival modulated incubation behavior with no effect on development. Hummingbirds had the highest egg surface-area to volume ratios among all birds; in 147 species, incubation periods increased with decreasing ambient temperatures. We propose a new hypothesis to explain how in cold conditions, heat transfer to the embryo is fragmented into smaller increments and therefore extended over longer periods, underscoring the role of adult behavior in mediating embryonic temperature mismatches and life-history tradeoffs.

ecology↗

bioRxiv2025

Characterization of the BRAF interactome identifies BRAFV600E<=>TP53 interaction in melanoma

Melanoma is a highly aggressive and frequently metastatic cancer with its incidence reported to be on the rise. Although most oncogenic drivers in melanoma converge on activation of the RAS>RAF>MEK>ERK MAPK signaling pathway, not all MAPK-activating mutations are recurrently observed in this disease, suggesting a unique functional role for BRAFV600E, which is present in [~]50% of all melanoma cases. However, the prevalence of BRAFV600E alterations over other known MAPK-promoting oncoproteins raises questions regarding whether BRAFV600E possesses additional functions outside of MAPK pathway activation. Thus, we performed TurboID to differentiate the interactome between wild-type BRAF and BRAFV600E. We identified novel interacting partners of normal vs. BRAFV600E, most strikingly being the tumor suppressor TP53. While TP53 is commonly altered or lost across many malignancies, it is notable that TP53 alterations are rare in melanoma. Our studies suggest that BRAFV600E can interact with and inactivate TP53, thus providing potential mechanistic explanation as to why TP53 inactivation or loss is infrequent in BRAFV600E-driven melanoma.

cancer biology↗

bioRxiv2025

On the onset of multicellular invasive behavior in hierarchical lineage: the role of inhibitory feedback and local fluctuations

The emergence of multicellular invasive behavior is a key characteristic of various biological processes, including wound healing, development, and tissue regeneration. In this study, we develop a lattice-gas cellular automaton (LGCA) model to explore the role of inhibitory feedback in the invasive behavior of a hierarchical lineage composed of stem cells and differentiated cells. We consider both non-spatial and spatial stochastic models to investigate how spatial interactions influence invasion dynamics. Our findings suggest that inhibitory feedback from differentiated cells significantly impacts the invasive potential of stem cells. In addition, local fluctuations induce unstable fronts that move with relatively low speed. Finally, we explore the implications of our work for understanding the regulation of multicellular dynamics in various pathophysiological contexts.

cancer biology↗

bioRxiv2025

Seasonal dynamics and thermal vulnerability of marine microbial communities in Uruguayan coastal waters

In an era of accelerating ocean warming, understanding the drivers of microbial community composition is more urgent than ever. Long-term time-series studies are particularly powerful for this purpose. However, most previous work remains confined to the Northern Hemisphere and focuses solely on taxonomy, leaving the Southern Hemisphere underrepresented and microorganisms functional dimensions underexplored. Moreover, phenomena such as current-driven community shifts and microbial vulnerability to warming remain poorly understood. Here, we address these gaps by analyzing microbial communities over two years at the South Atlantic Microbial Observatory (SAMO), located off the coast of Uruguay within a Marine Protected Area. SAMO, situated under the contrasting influences of the Brazil and Malvinas currents and recognized as a global-warming hotspot, provides an ideal setting to study the temporal dynamics of marine microbial communities. Using shotgun metagenomics and 16S rRNA amplicon sequencing, we revealed strong seasonal shifts driven by environmental variability and the influence of these two ocean currents, resulting in two community states (summer-autumn vs winter-spring). These states exhibited contrasting composition, diversity, and assembly mechanisms. Further, our results show that taxonomic and functional diversity trends diverge between these periods. Crucially, our work suggests that the predicted tropicalization of Uruguays coast is likely to boost taxonomic richness but erode functional repertoires, while also increasing vulnerability to ocean warming, potentially undermining biogeochemical functioning.

microbiology↗

bioRxiv2025

High-Throughput Multiomics Profiling of Model Systems Using the AVITI24 Platform

We present a multiomics platform comprising Teton, a detection assay system, and AVITI24, a dual-flowcell instrument that performs both cellular imaging and sequencing readout. Teton integrates a compartmentalized flowcell for cell culture with methods to measure morphology, RNA, and protein at subcellular resolution. The platform quantifies morphological features through cell painting of 6 cellular components, RNA expression of up to 350 transcripts via sequencing of oligonucleotides hybridized to mRNA, and protein expression of up to 200 targets using antibody-linked oligonucleotide sequencing. The flow cell accommodates >1 million cells in a 10 cm squared open-well format or can be subdivided into 12 or 48 wells to support experiments with multiple conditions or time points. We describe and validate the detection methods of the platform and showcase its capabilities by co-culturing three cancer cell lines and elucidating the cellular pathways triggered by various drug treatments as a function of time. Using multiple time points enables us to capture the dynamics of cellular processes including receptor activation and signaling cascades. The results demonstrate how different cancer cells evade TNF-induced apoptosis by activating compensatory signaling programs that maintain survival despite pro-apoptotic cues. Our model system replicates previously published results and highlights the versatility of the platform in enabling rapid, high-throughput analysis of complex cellular responses in varied biological contexts.

genomics↗

bioRxiv2025

Harmonized Protocol for Subfield Segmentation in the Hippocampal Body on High-Resolution in vivo MRI from the Hippocampal Subfields Group (HSG)

Hippocampal subfields differentially develop and age, and they vary in vulnerability to neurodegenerative diseases. Innovation in high-resolution imaging has accelerated clinical research on human hippocampal subfields, but substantial differences in segmentation protocols impede comparisons of results across laboratories. The Hippocampal Subfields Group (HSG) is an international organization seeking to address this issue by developing a histologically-valid, reliable, and freely available segmentation protocol for high-resolution T2-weighted 3 tesla MRI (http://www.hippocampalsubfields.com). Here, we report the first portion of the protocol focused on subfields in the hippocampal body; protocols for the head and tail are in development. The body protocol includes definitions of the internal boundaries between subiculum, Cornu Ammonis (CA) 1-3 subfields, and dentate gyrus, in addition to the external boundaries of the hippocampus apart from surrounding white matter and cerebrospinal fluid. The segmentation protocol is based on a novel histological reference data set labeled by multiple expert neuroanatomists. With broad participation of the research community, we voted on the segmentation protocol via online survey, which included detailed protocol information, feasibility testing, demonstration videos, example segmentations, and labeled histology. All boundary definitions were rated as having high clarity and reached consensus agreement by Delphi procedure. The harmonized body protocol yielded high inter- and intra-rater reliability. In the present paper we report the procedures to develop and test the protocol, as well as the detailed procedures for manual segmentation using the harmonized protocol. The harmonized protocol will significantly facilitate cross-study comparisons and provide increased insight into the structure and function of hippocampal subfields across the lifespan and in neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Mitochondrial structure despite nuclear panmixia: sex-specific dispersal dictates population structure in sperm whales

Marine mammals have high potential for dispersal, yet behavioral or environmental constraints can limit gene flow. This is true for the endangered sperm whale, Physeter macrocephalus, which has a global distribution and long-distance migrations. While previous studies revealed mitochondrial population structure with weak nuclear structure globally, genomic approaches examining this pattern have been limited. Understanding connectivity is critical for the management of this species due to population declines relative to pre-whaling, recent impacts from the Deepwater Horizon oil spill, and ongoing threats from anthropogenic sound. We investigated the connectivity between two regions, the U.S. Gulf of America (Mexico) and western North Atlantic Ocean, using reduced representation genomics and mitochondrial control region sequencing of 73 sperm whales. We found that relatedness decreased with geographic distance, likely due to the presence of familial structure. Nuclear markers showed no population structure (FST = 0.001-0.008), while mitochondrial structure was high (FST = 0.36-0.65), consistent with male-biased dispersal and female philopatry. Across all samples, genetic diversity (nuclear: 0.0014; mitochondrial: 0.0017) and effective population size (Ne=460) were low. Given this low diversity and evidence for the partitioning of genetic variation, we recommend managers treat these two regions as distinct in order to preserve existing variation and promote resilience of this species. These results show that despite the increased power of a genomic approach, it is essential to consider the biology of the species at hand and leverage both mitochondrial and nuclear markers to understand the genetic structure of threatened species.

evolutionary biology↗

bioRxiv2025

Development and evaluation of screwworm, Cochliomyia hominivorax, transgenic sexing strains with embryonic gene promoters for a genetic control program

The New World screwworm, Cochliomyia hominivorax, was eradicated from North and Central America through the first application of the sterile insect technique. The sterile screwworm adult fly releases were mixed sex, but experience with other flies suggests that releasing only males could be up to five times more effective. Here we describe screwworm transgenic sexing strains (TSSs) with expected embryo lethality developed using the Tetracycline-off (Tet-off) system with the Lucilia cuprina nullo (DR6) or C. macellaria CG14427 (DR7) gene promoters and a tTA activated effector to promote female lethality. The TSSs expressed tTA highest in 2-3 h embryos and low in larvae and adults. However, most strains showed high expression in pupae. Evaluation on two doxycycline (Dox) regimens found that inclusion of Dox in the last larval feeding rescued females of the subsequent generation, likely by maternal Dox transfer to embryos. All TSSs produced only males on a reduced Dox feeding regimen but the female lethal period for the DR6 TSS was too late in development to save diet costs. Production parameters were met by all strains in colony, but strains had lower male fly survival than current production strains after removing Dox. In non-competitive mating success trials DR6 strains performed poorly, but DR7 performed equally to production strain males. However, males from all TSSs faired poorly in mating competition tests against production males. Our study highlights the importance of tightly-regulated gene promoters and suitable antibotics feeding schemes for the development and evaluation of TSSs based on Tet-off system.

biochemistry↗

bioRxiv2025

Ophiobolin A selectively alters mitochondria, metabolism and redox biology in breast cancer and mammary epithelial cells which have undergone epithelial to mesenchymal transition

Breast cancer progression is facilitated by the epithelial to mesenchymal transition (EMT), generating cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics. EMT is known to impart changes in metabolic pathways and mitochondrial function. Here, we show a natural product possesses EMT-specific cytotoxic activity via alterations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, although its target and mechanism of action remain unknown. Here, we utilized a model of mammary epithelial cells and breast cancer cell lines with and without EMT features to characterize the mechanism of selectivity towards EMT(+) cells by OpA and to identify novel targets for the treatment of EMT-enriched breast cancer. Proteins interacting with OpA in EMT(+) cells were identified through proteomic studies. We utilized trans-mitochondrial cybrids to determine that mitochondria mediate sensitivity to OpA. Furthermore, we report effects on glycolysis, oxidative metabolism, and disruption of metabolite abundance in the TCA cycle. Alterations to mitochondrial iron concentrations and glutathione production, mediated partly by OpA engagement with the metabolic proteins CISD3 and SLC25A40, were also detected in EMT(+) cells. Antioxidant mechanisms are activated by OpA in EMT(+) cells via the NRF2-ARE pathway, verified by decreased cytotoxicity in EMT(+) cells pretreated with the NRF2 activator CDDO. Collectively, we conclude that OpA selectivity toward EMT is mediated by the mitochondria, and at sub-cytotoxic levels, generates a metabolic shift leading to cell death countered by antioxidant mechanisms.

cancer biology↗

bioRxiv2025

Single-cell-guided identification of logic-gated antigen combinations for designing effective and safe CAR therapy

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies. However, its application in solid tumors remains limited because single targets are unlikely to suffice due to tumor antigen heterogeneity and off-tumor toxicities. To overcome these obstacles, we developed LogiCAR designer, a computational approach that utilizes single-cell transcriptomics data from patient tumors to systematically identify the cancer-specific antigen circuits with logic gates ("AND," "OR," and "NOT") that target the majority of cancer cells in a tumor while sparing normal cells and tissues as much as possible. LogiCAR designer efficiently scales to higher-order antigen combinations involving up to five genes. Applied to a large-scale dataset encompassing approximately 2 million cells (including > 620k tumor cells) from 342 clinical patient samples across all major breast cancer subtypes, LogiCAR designer identified antigen circuits with enhanced tumor-targeting efficacy and improved safety profiles compared to both previously reported circuits and single-target therapies in clinical trials. However, even these optimized shared circuits still proved insufficient for some patients. We hence systematically studied LogiCAR designers ability to identify highly effective CAR circuits that are individualized to each patient. Remarkably, such personalized CAR circuits provide estimated tumor-targeting efficacy tantamount to complete response in 76% of patients and partial response for all patients. Taken together, this analysis is the first systematic quantification of the efficacy and safety of all possible CAR circuits, showing that: (a) the quality of existing solutions leaves much to be desired; (b) the ability of shared circuits optimized across many patients is moderate, and finally, (c) individually tailored circuits offer significantly higher tumor-targeting efficacies for patients. LogiCAR designer offers a rigorous, data-driven way to facilitate the rational design of safe and effective CAR-based immunotherapies for cancer. Statement of Significance Development of a computational approach that efficiently identifies logic-gated CAR target combinations, called circuits, from single-cell transcriptomics, addressing a critical unmet clinical need. Application to the largest ensemble of breast cancer datasets to date, comprising [~]2 million cells (> 620k tumor cells) from 17 clinical cohorts, to identify CAR circuits predicted to be effective. Comprehensive safety profiling of candidate circuits spanning major tissues at both RNA and protein levels. Logic-gated CAR circuits generated by our pipeline address tumor heterogeneity and achieve efficacy and safety scores that surpass clinical trial and previously computationally identified circuits. Individualized rational CAR design offers a transformative approach to deliver precision-engineered CAR therapies with unprecedented efficacy.

bioinformatics↗

bioRxiv2025

Mechanical strain modulates Min patterning and division in E. coli filaments

Bacteria often encounter physico-chemical stresses that disrupt division, leading to filamentation, where cells elongate without dividing. While this adaptive response enhances survival, it also exposes filaments to significant mechanical strain, raising questions about the mechanochemical feedback in bacterial systems. In this study, we investigate how mechanical strain influences the Min oscillatory system, a reaction-diffusion network central to division in Escherichia coli. Through a multidisciplinary approach combining quantitative fluorescence microscopy, biophysical modeling, microfluidics, and patterned growth substrates, we demonstrate that filamentous E. coli undergoes growth-induced buckling instability. This phenomenon alters the diffusivity of membrane proteins and modulates the spatiotemporal patterning of the Min system. Moreover, we show that this mechanochemical interplay determines division site positioning after stress relief, effectively creating a mechanical "memory" for cytokinesis. Our findings underscore the critical role of mechanical forces in bacterial filamentation and provide new insights into the functional implications of mechanobiology in microbial systems.

biophysics↗

bioRxiv2025

Macroinvertebrate diversity patterns in a guano-rich temperate cave

Most caves in temperate regions are energy-poor, leading to low richness and abundances of terrestrial macroinvertebrates. However, caves with abundant bat populations may represent a notable exception, as bat guano serves as a significant source of organic matter, supporting diverse specialized invertebrates. Yet, given the overall rarity of guano-rich caves in temperate region, their ecological dynamics are poorly understood. We conducted a year-long monthly standardized monitoring of the macroinvertebrates of the Buca dei Ladri cave, a guano-rich cavity in Tuscany, Italy. At each survey, we counted species within different sampling quadrats and collected environmental variables such as temperature, structural heterogeneity of the substrate, dominant substrate, and presence of bats. Over the year, we identified 31 macroinvertebrate species in the cave as both morphospecies and molecular lineages, based on mitochondrial COI and nuclear 18S genes. The latter approach hinted at the presence of cryptic diversity in some species. We observed an increase in species diversity with increasing structural complexity and the presence of bats, whereas a lower species diversity was found in guano-dominated substrates, probably due to the old age and subsequent low nutritional value of the historical guano deposits. Changes in the abundance of species within the cave were primarily explained by differences in structural complexity across sampling quadrats, whereas the distance from the cave entrance mostly explained species replacement. Our results underscore the importance of preserving healthy bat populations and a high habitat heterogeneity within the cave for the long-term conservation of the unique macroinvertebrate assemblages of the cave.

ecology↗

bioRxiv2025

Mechanism of forced-copy-choice RNA recombination by enteroviral RNA-dependent RNA polymerases

Forced-copy-choice recombination occurs at the end of a template, differing from copy-choice recombination, which happens at internal positions. This mechanism may produce full-length genomes from fragments created by host antiviral responses. Previous studies from our laboratory demonstrated that poliovirus (PV) RNA-dependent RNA polymerase (RdRp) switches to an "acceptor" template in vitro when initiated on a heteropolymeric RNA-primed "donor" template. Surprisingly, recombinants showed template switching from the 3-end of the donor template. We have developed a primed-template system to study PV RdRp-catalyzed forced-copy-choice RNA recombination. PV RdRp adds a single, non-templated nucleotide to the 3-end of a blunt-ended, double-stranded RNA product, forming a "plus-one" intermediate essential for template switching. Non-templated addition of CMP was favored over AMP and GMP (80:20:1); UMP addition was negligible. A single basepair between the plus-one intermediate and the 3-end of the acceptor template was necessary and sufficient for template switching, which could occur without RdRp dissociation. Formation of the plus-one intermediate was rate limiting for template switching. PV RdRp also utilized synthetic, preformed intermediates, including those with UMP 3-overhangs. Reactions showed up to five consecutive template-switching events, consistent with a repair function for this form of recombination. PV RdRp may exclude UMP during forced-copy-choice RNA recombination to preclude creation of nonsense mutations during RNA fragment assembly. Several other picornaviral RdRps were evaluated, and all were capable of RNA fragment assembly to some extent. Lastly, we propose a structure-based hypothesis for the PV RdRp-plus-one intermediate complex based on an elongating PV RdRp structure.

biochemistry↗

bioRxiv2025

Distal mutations in a designed retro-aldolase alter loop dynamics to shift and accelerate the rate-limiting step

Amino-acid residues distant from an enzymes active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

biochemistry↗

bioRxiv2024

Pharmacological inhibition of SUMOylation recapitulates genetic hypoSUMOylation in the murine epididymal white adipose tissue

Post-translational modification by the small ubiquitin-like modifier (SUMO) is essential for cellular differentiation and homeostasis. Here, we investigate the role of SUMOylation in adipose tissue development using TAK-981, a pharmacological inhibitor of SUMOylation. Administration of TAK-981 to mice resulted in significant defect in weight gain and adipocyte atrophy in perigonadal white adipose tissue (gWAT) depots. Gene expression analyses revealed a marked downregulation of adipogenic genes, including Pparg, Cebpa, and Fasn. Our data thus indicate that TAK-981 treatment impaired adipogenesis in gWAT, consistent with prior findings that SUMOylation supports transcriptional regulation of adipogenesis and lipid metabolism. We also found significant infiltration of immune cells and efferocytosis in gWAT. Our results thus indicate that SUMOylation inhibition using a small molecule phenocopies genetic hypoSUMOylation models, highlighting its critical role in maintaining adipocyte functionality and immune environment. These findings provide evidence that SUMOylation is essential for fat accumulation in vivo. Furthermore, given that TAK-981 is currently under clinical evaluation for the treatment of solid tumors, our results underscore the importance of considering the potential unintended effects of SUMOylation inhibition on adipose tissue in patients.

developmental biology↗

bioRxiv2024

Quantification of subcellular RNA localization through direct detection of RNA oxidation

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, nucleus, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for the analysis of localized RNAs across different biological systems.

molecular biology↗