Biology subjects

Pelechano, V.

Publications and source records attributed to Pelechano, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Balancing spatial resolution and proteome depth in LC-MS based spatialproteomics

Spatial proteomics aims to resolve protein composition within intact tissues, yet extraction-based liquid chromatography-mass spectrometry (LC-MS) workflows face an inherent trade-off: smaller sampling units increase spatial specificity, whereas larger sampling units provide greater proteome depth and robustness. As analytical sensitivity improves, sampling-unit size therefore becomes a key experimental design parameter. Current extraction-based LC-MS workflows typically rely on laser capture microdissection (LCM), where sample recovery and scalability can become limiting at low input. Spatially resolved laser-activated cell sorting (SLACS) offers an alternative tissue-isolation strategy based on single-pulse near-infrared laser activation. Here, we use SLACS to systematically examine the resolution-sensitivity trade-off across sampling units ranging from single-cell-equivalent to larger low-input tissue regions. Few-cell sampling retained substantial proteomic information relative to larger regions while increasing spatial specificity. Applied to the mouse somatosensory cortex, SLACS generated deep, layer-resolved proteomic profiles from regions corresponding to approximately 60 cells and preserved major layer-specific molecular patterns at inputs as low as approximately 6 cells. These results highlight sampling-unit size as an important experimental design parameter in extraction-based spatial proteomics and support few-cell sampling as a practical compromise between spatial specificity, proteome depth and robustness.

biochemistry↗

bioRxiv2026

Identifying cancer cell-state transitions from multimodal single-cell data

Phenotypic plasticity allows cancer cells to evade therapy, yet the transient nature of state transitions has made their molecular drivers difficult to define. Here, we present a single-cell framework that leverages the temporal delays between mRNA and protein accumulation to directly capture cells undergoing phenotypic switching. We show that differentiation-associated delays between transcript and protein accumulation are detectable in multimodal single-cell data as discordant mRNA and surface-protein levels. Applying this strategy to the K562 leukemia model, which alternates between differentiated CD24- and progenitor-like CD24+ states, we identify transitioning cells and derive a transcriptional signature linking cell-cycle progression and mitochondrial remodeling to plasticity. Genome-wide CRISPR screening confirms key regulators of plasticity, including BCR-ABL1 and mitochondrial homeostasis genes. We summarize the transition-associated program into a score that predicts imatinib response in chronic myeloid leukemia, stratifies survival in acute myeloid leukemia, and retains prognostic value across 31 TCGA tumor types. Spatial transcriptomics reveals localized plasticity hotspots in solid tumors. Together, this framework exposes the molecular basis of cancer plasticity and enables its quantification across tumors.

cancer biology↗

bioRxiv2026

Puf3 contributes to changes in mRNA solubility, translation elongation dynamics at rare arginine codons and loss of protein homeostasis in cells lacking Not4

The Not proteins of the Ccr4-Not complex regulate translation elongation dynamics, essential for proper folding and assembly of new proteins. In yeast, ribosomes with non-optimal codons in the A-site are enriched within the pool of ribosomes bound by Not4 and Not5. Such ribosomes accumulate in cells lacking Not4 or Not5 that show defects in co-translational assembly and aggregation of new proteins. Recently we observed that depletion of Not1 and Not4 inversely regulate changes in mRNA solubility, correlating with inverse codon-specific changes in A-site ribosome dwelling occupancies (RDOs). Here we describe that mRNAs less soluble upon Not4 depletion are enriched for targets of the RNA-binding protein Puf3. We determine that Puf3 contributes to inverse changes of A-site RDOs upon Not1 and Not4 depletion, in particular at rare arginine codons, and it contributes to changes in mRNA solubility in not4{Delta}. Moreover, deletion of Puf3 suppresses temperature sensitivity and protein aggregation in the not4{Delta} strain, while overexpression of Puf3 is toxic. Puf3 post-translational modifications and the Puf3 interactome are altered in not4{Delta}. Taken together, our results associate alterations in Puf3 post-translational status and function, including contribution to translation elongation dynamics, with not4{Delta} mutant phenotypes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/695943v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@855afdorg.highwire.dtl.DTLVardef@118addaorg.highwire.dtl.DTLVardef@13dd015org.highwire.dtl.DTLVardef@12824c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A streamlined nanopore-compatible 5PSeq protocol for rapid phenotypic antimicrobial sensitivity testing

Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present "simplified 5PSeq" (s5PSeq), a streamlined protocol for profiling 5 monophosphorylated (5P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition--offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics. HighlightsO_LIs5PSeq is a streamlined protocol for profiling 5P mRNA degradation intermediates. C_LIO_LIContext-specific ribosome stalls can be used to assess phenotypic antimicrobial sensitivity at the molecular level. C_LIO_LIBlocking rRNA sequencing at the ligation step streamlines library preparation and lowers costs and hands-on time. C_LIO_LIIntegration with nanopore sequencing allows same-day antimicrobial sensitivity testing in species with 5-3 exonuclease. C_LI

microbiology↗

bioRxiv2024

XRN1 supplies free nucleotides to feed alphavirus replication

Several RNA viruses induce widespread degradation of cellular mRNAs upon infection; however, the biological significance and mechanistic details of this phenomenon remain unknown. Here, we make use of a model alphavirus, Sindbis virus (SINV), to fill this knowledge gap. We found that SINV triggers cellular RNA decay through the exonuclease XRN1 and the 5-to-3 degradation machinery (5-3DM). These proteins accumulate at viral replication organelles (VROs) and interact with the non-structural protein 1 (nsP1), bringing mRNA degradation into proximity with vRNA synthesis. Our data suggest that monophosphate nucleotides released by cellular RNA decay are recycled through the salvage pathway to feed viral replications. Our work thus reveals a fundamental connection between cellular mRNA degradation and viral replication via nucleotides repurposing. Research highlightsO_LI5-3 RNA decay is essential for the replication of a wide range of viruses. C_LIO_LIXRN1 directly interacts with transcripts which are degraded during infection. C_LIO_LIRNA decay factors and salvage pathway members localise to viral factories. C_LIO_LISupplying nucleosides to several 5-3DM deficient cells facilitates SINV infection. C_LI

biochemistry↗

bioRxiv2024

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys> isoacceptors in the P site

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

molecular biology↗

bioRxiv2024

Ribosomes modulate transcriptome abundance via generalized frameshift and out-of-frame mRNA decay.

Cells need to adapt their transcriptome to quickly match cellular needs in changing environments. mRNA abundance can be controlled by altering both its synthesis and decay. Here we show how, in response to poor nutritional conditions, the bulk of the S. cerevisiae transcriptome undergoes -1 ribosome frameshifts and experiences an accelerated out-of-frame co-translational mRNA decay. Using RNA metabolic labelling, we demonstrate that in poor nutritional conditions, NMD-dependent degradation represents at least one third of the total mRNA decay. We further characterize this mechanism and identify low codon optimality as a key factor for ribosomes to induce out-of-frame mRNA decay. Finally, we show that this phenomenon is conserved from bacteria to humans. Our work provides evidence for a direct regulatory feedback mechanism coupling protein demand with the control of mRNA abundance to limit cellular growth and expands the functional role of mRNA quality control.

molecular biology↗

bioRxiv2023

The exosome degrades chromatin-associated RNAs genome-wide and maintains chromatin homeostasis

Chromatin-associated RNAs (caRNAs) modulate chromatin organization and function. The RNA exosome degrades different types of nuclear transcripts, but its role in chromatin has not been addressed. Here we have used Drosophila melanogaster S2 cells as a model system to identify the repertoire of caRNAs and establish the role of the exosome in their regulation. We have analyzed both unique and repetitive sequences, and combining RNA-seq and ATAC-seq we show that the simultaneous depletion of the exosome catalytic subunits RRP6 and DIS3 not only affects caRNA levels but also changes the local chromatin accessibility at specific loci. We have identified a group of exosome-sensitive genes that are involved in developmental regulation and are characterized by a balanced chromatin state in which Polycomb and Trithorax factors coexist. Our results reveal that RNA degradation by the exosome is an important mechanism for the homeostasis of such balanced chromatin states. Given that eukaryotic genomes are repetitive to a large extent, we have also analyzed repetitive caRNAs (rep-caRNAs) and we show that the exosome is needed to control repcaRNA levels and to maintain the degree of chromatin packaging in repetitive genomic regions. This role is particularly relevant in the pericentromeric regions where the exosome is required to silence LTR elements and maintain centromere organization.

genomics↗

bioRxiv2023

Cellular energy regulates mRNA translation and degradation in a codon-specific manner

BackgroundCodon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. ResultsHere we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Biochemical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly affect the decoding kinetics of different codons. ConclusionsThis new mechanism of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression.

systems biology↗

bioRxiv2023

The association of the RSC remodeler complex with chromatin depends on prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin to gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely to be arrested or slower. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

molecular biology↗

bioRxiv2022

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation

Cancer cells outgrowing in distant organs of metastasis rewire their metabolism to fuel on the available nutrients. While this is often considered an adaptive pressure limiting metastasis formation, some nutrients available at the metastatic site naturally or through changes in organ physiology may inherently promote metastatic growth. We find that the lung, a frequent site of metastasis, is a lipid-rich environment. Moreover, we observe that pathological conditions such as pre-metastatic niche formation and obesity further increase the availability of the fatty acid palmitate in the lung. We find that targeting palmitate processing inhibits spheroid growth in vitro and metastasis formation in lean and obese mice. Mechanistically, we discover that breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a (CPT1a)-dependent manner. Lysine acetyltransferase 2a (KAT2a), whose expression is promoted by palmitate availability, relies on the available acetyl-CoA to acetylate the NF-{kappa}B subunit p65. This favors nuclear location of p65 and activates a pro-metastatic transcriptional program. Accordingly, deletion of KAT2a phenocopies CPT1a silencing in vitro as well as in vivo and patients with breast cancer show co-expression of both proteins in metastases across palmitate-rich metastatic sites. In conclusion, we find that palmitate-rich environments foster metastasis growth by increasing p65 acetylation resulting in elevated NF-{kappa}B signaling.

cancer biology↗

bioRxiv2022

Barcode-free prediction of cell lineages from scRNA-seq datasets

The integration of lineage tracing with scRNA-seq has transformed our understanding of gene expression heritability during development, regeneration, and disease. However, lineage tracing is technically demanding and most existing scRNA-seq datasets are devoid of lineage information. Here we introduce Gene Expression Memory-based Lineage Inference (GEMLI), a computational pipeline allowing to predict cell lineages over several cell divisions solely from scRNA-seq datasets. GEMLI leverages genes displaying conserved expression levels over cell divisions, and allows i.a. identifying cell lineages in a broad range of cultured cell types, in intestinal organoids, and in crypts from adult mice. GEMLI recovers GO-terms enriched for heritable gene expression, allows to discriminate symmetric and asymmetric cell fate decisions and to reconstruct individual cellular structures from pooled scRNA-seq datasets. GEMLI considerably extends the pool of datasets from which lineage information can be obtained, thereby facilitating the study of gene expression heritability in a broad range of contexts. GEMLI is available at (https://github.com/UPSUTER/GEMLI).

developmental biology↗

bioRxiv2022

Differential regulation of mRNA stability modulates transcriptional memory and facilitates environmental adaptation

Transcriptional memory, by which cells respond faster to repeated stimuli, is key for cellular adaptation and organism survival. Factors related to chromatin organization and activation of transcription have been shown to play a role in the faster response of those cells previously exposed to a stimulus (primed). However, the contribution of post-transcriptional regulation is not yet explored. Here, combining flow cytometry and high throughput sequencing, we perform a genome-wide screen to identify novel factors modulating transcriptional memory in S. cerevisiae in response to galactose nutrition sources. In addition to the well-known chromatin factors modulating transcriptional memory, we find that depletion of the nuclear RNA exosome increases GAL1 expression in primed cells. We perform a genome-wide characterisation of this process and show that changes in nuclear surveillance factor association can enhance both gene induction and repression in primed cells. Finally, we show that in addition to nuclear mRNA degradation, differences in cytoplasmic mRNA decay also modulate transcriptional memory and contribute to faster gene expression remodelling in primed cells. Our results demonstrate that mRNA post-transcriptional regulation, and not only transcription regulation, should be considered when investigating gene expression memory.

genomics↗

bioRxiv2022

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

BackgroundThe Ccr4-Not complex is most well known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. ResultsIn this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impact mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. ConclusionOur results reveal that mRNA solubility defines dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

molecular biology↗

bioRxiv2021

RNA degradation analysis reveals ribosome dynamics in complex microbiome samples

Post-transcriptional regulation is essential for life, yet we are currently unable to investigate its role in complex microbiome samples. Here we discover that co-translational mRNA degradation, where the degradation machinery follows the last translating ribosome, is conserved across prokaryotes. By investigating 5P mRNA decay intermediates, we obtain in vivo ribosome protection information that allows the study of codon and gene specific ribosome stalling in response to stress and drug treatment at single nucleotide resolution. We use this approach to investigate in vivo species-specific ribosome footprints of clinical and environmental microbiomes and show for the first time that ribosome protection patterns can be used to phenotype microbiome perturbations. Our work paves the way for the study of the metatranslatome, and enables the investigation of fast, species-specific, post-transcriptional responses to environmental and chemical perturbations in unculturable microbial communities.

genomics↗

bioRxiv2020

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

BackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

genomics↗

bioRxiv2020

Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast

Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

molecular biology↗

bioRxiv2020

High-throughput 5'P sequencing reveals environmental regulated ribosome stalls at termination level

RNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is connected to the translation process up to the degree that 5-3 mRNA degradation follows the las translating ribosome. Here we present an improved high-throughput 5P degradome RNA sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable and uses affordable duplex-specific nuclease based rRNA depletion. We investigate in vivo ribosome stalls focusing on translation termination. By comparing ribosome stalls identified by ribosome profiling, disome-seq and HT-5PSeq we identify that degradation-associated ribosome stalls are often enriched in Arg preceding the stop codon. On the contrary, mRNAs depleted for those stalls use more frequently TAA stop codon preceded by hydrophobic amino acids. Finally, we shown that termination stalls identified by HT-5Pseq, and not by other approaches, are associated to decreased mRNA stability. Our work suggests that ribosome stalls associated to mRNA decay can be easily captured by investigating the 5P degradome.

genomics↗