Biology subjects

da Cunha, J. P.

Publications and source records attributed to da Cunha, J. P..

8 recordsLinked to original sources

bioRxiv2026

Phosphorylation patterns of pre-ribosomal proteins associated with the RNA exosome

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction, although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co-immunoprecipitation followed by phosphopeptide enrichment and high-resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co-immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insights into the posttranscriptional control of gene expression.

molecular biology↗

bioRxiv2025

First Genome-Wide Centromere Map of Trypanosoma cruzi Reveals Linear and 3D Compartment Boundaries and Spatial Clustering

Background: Trypanosoma cruzi, the etiological agent of Chagas disease, possesses a highly repetitive genome that has historically hindered high-quality assembly and structural characterization. Despite significant advances in assembling T. cruzi genomes, major gaps remain. Among these, the complete repertoire of centromeric sequences has remained elusive, representing a critical missing piece in our understanding of chromosome structure and inheritance. Results: Here, we generated high-coverage Hi-C (genome-wide chromosome conformation capture) data for the widely used T. cruzi Dm28c strain improving its genome assembly, reducing the number of scaffolds and producing a more contiguous and accurate genome. To investigate centromere organization, we performed ChIP-seq using the mNeonGreen-myc-tagged kinetochore proteins KKT2 and KKT3, resulting in the identification of 40 KKT-enriched peaks across 29 scaffolds. These peaks were located in regions enriched in retrotransposable elements, particularly L1Tc and VIPER, near strand switch regions, areas of high GC content, and at the boundaries between conserved genes and virulence-factor multigene families. Conclusion: Notably, Hi-C analysis revealed that centromeres may act as structural boundaries contributing to genome compartmentalization and frequently engage in 3D spatial clustering, suggesting a role in higher-order nuclear architecture. Overall, our study provides a high-quality reference genome for the Dm28c strain, presents the first genome-wide centromere map in T. cruzi, and offers novel insights into centromere-mediated 3D genome organization

genomics↗

bioRxiv2025

Nuclear proteome analysis reveals spatial and temporal compartmentalization of metabolic enzymes during Trypanosoma cruzi cell cycle

Trypanosoma cruzi, the etiological agent of Chagas Disease (CD), represents a significant public health concern and serves as a valuable model for investigating the cell cycle in early-diverging eukaryotes. Its unique cellular features--including the absence of chromosome condensation and the coordination of nuclear division with specialized organelles--provide insights into non-canonical regulatory mechanisms, and underscore the pivotal role of the nucleus in maintaining genomic integrity and regulating fundamental processes such as DNA replication and gene expression. To investigate nuclear dynamics throughout the cell cycle, we synchronized T. cruzi epimastigotes at G1/S, S, and G2/M using hydroxyurea (HU), and isolated nuclear proteins were quantitative evaluated by LC-MS/MS. We identified 2,937 nuclear-enriched proteins, revealing distinct, phase-specific expression patterns. The G1/S phase was marked by increased levels of metabolic enzymes including those related to energy and nucleotide/nucleoside metabolisms. The S phase showed elevated abundance of canonical and variant histones, consistent with chromatin remodeling and DNA replication. The G2/M phase was enriched in proteins involved in protein synthesis and microtubule dynamics, essential for mitosis. Notably, [~]6% of the nuclear proteome consisted of metabolic enzymes, a finding further supported by other public proteomics datasets and in vitro nuclear activity assays. These results suggest that metabolic enzymes and/or their metabolites may modulate nuclear processes in T. cruzi, potentially influencing the epigenome and gene expression regulation, as proposed in other eukaryotic models. Altogether, our findings reveal dynamic remodeling of the nuclear proteome during the T. cruzi cell cycle and point to a previously underappreciated role for metabolic enzymes likely regulating nuclear functions in a cell cycle-dependent manner.

microbiology↗

bioRxiv2025

Remodeling of tRNA modification in Trypanosoma cruzi life forms

Trypanosoma cruzi, the etiological agent of Chagas disease, infects millions of people in the Americas. This parasite undergoes drastic changes in its morphology and metabolism between infective and noninfective forms through global remodeling of its proteome. Chemical modification of tRNA (tRNA modification) contributes to the control of protein expression by modulating the codon decoding process. However, knowledge of tRNA modification profiles, the enzymes that create modifications and their regulation in different cellular conditions is largely restricted to relatively few model organisms. Here, we profile tRNA modifications in both infective and noninfective forms of T. cruzi to probe their dynamic changes. Genome mining of tRNA modifying enzymes identified 66 putative tRNA modifying enzymes in T. cruzi, each responsible for at least one of fifty-seven modifications. tRNA sequencing detected reverse transcription-derived signatures at 182 sites in T. cruzi tRNAs that are likely derived from 18 tRNA modifications. tRNA modifications and tRNA modification enzymes are differentially modulated across the life stages of T. cruzi. We found that hydroxywybutosine (OHyW) at position 37 on tRNAPhe had a reduced level in the infective form (metacyclic trypomastigote) and the associated modification enzyme Tyw1a exhibited reduced expression in this stage. Knockout of Tyw1a increased the differentiation from epimastigote (noninfective form) to metacyclic trypomastigote, suggesting that OHyW37 modification levels control the rate of metacyclogenesis. Overall, our findings suggest that the global regulation of tRNA modifications in the life stages of T. cruzi plays a critical role in the differentiation of this parasite.

molecular biology↗

bioRxiv2025

Mass Spectrometry-Based Characterization of Histone Post-Translational Modifications in Sugarcane

Histones, the core components of nucleosomes, play an important role in chromatin structure and function through post-translational modifications (PTMs) that regulate gene expression without altering the DNA sequence. Sugarcane (Saccharum spp.), a highly polyploid and economically significant crop, has a complex genome with a limited understanding of histone PTMs. In this study, we employed mass spectrometry to characterise the PTMs of histones in internodes of field-grown commercial sugarcane, harvested at different times of the day, with a focus on histones H2A and H2B, which had not been previously studied in this context. Using a sorghum histone catalog as a reference, we identified 26 H1, 91 H2A, 70 H2B, 5 CENH3, 36 H3.1, 23 H3.3, 18 H3.3L, and 57 H4 loci in the sugarcane genome. We detected extensive PTMs, including acetylation and methylation, across histones H2A, H2B, H3, and H4. Canonical and variant histones showed distinct post-translational modification (PTM) patterns, highlighting their functional specialisation. This study provides a comprehensive catalog of sugarcane histone PTMs, offering insights into their roles in chromatin dynamics and gene regulation, with potential applications in crop improvement and stress adaptation.

plant biology↗

bioRxiv2024

Maspin/SerpinB5 is a cytoskeleton-binding protein that regulates epithelial cell shape

Maspin/SerpinB5 is an abundant and pleiotropic protein mostly expressed by epithelia. Initially described as a tumor suppressor, it has been reported as a regulator of cell adhesion, migration, and invasion. How intracellular Maspin orchestrates these processes is poorly understood. In this study, we utilized Affinity purification-Mass spectrometry (AP/MS) alongside in vitro reconstitution assays to establish that Maspin directly interacts with microtubules and microfilaments. Additionally, CRISPR/Cas9-mediated GFP tagging of endogenous Maspin, combined with immunostaining, revealed its localization at the cortical cytoskeleton and the mitotic spindle. Depletion of Maspin by RNAi and CRISPR/Cas9 in three distinct epithelial cell lines disrupts cell-cell adhesion, reorganizes the cytoskeleton and results in upregulation of mesenchymal markers during interphase. In mitotic cells, loss of Maspin induces abnormal cell rounding and rearrangement of cortical F-actin. Moreover, Maspin suppresses microtubule growth in vitro and in cells. Collectively, these results demonstrate that Maspin acts at the interface between the cytoskeleton and adhesion sites, directly modulating cell shape and preventing epithelial-mesenchymal transition. SummaryDa Silva et al. report that the non-inhibitory serpin Maspin (SerpinB5), which has long been implicated in the regulation of cell adhesion, migration, invasion and metastasis, directly binds to microfilaments and microtubules in vitro and in cells, acting at the interface between the cytoskeleton and adhesion sites.

cell biology↗

bioRxiv2024

Integrating tDNA Epigenomics and Expression with Codon Usage Unravel an Intricate Connection with Protein Expression Dynamics in Trypanosoma cruzi

Codon usage bias impacts protein expression across all kingdoms of life, including trypanosomatids. These protozoa, such as the Trypanosoma cruzi, primarily regulate their protein-coding genes through posttranscriptional mechanisms. Here, we integrated analyses of codon usage with multiple high-throughput sequencing data to investigate whether codon usage bias is present into surface virulence factors (disruptive compartment), conserved housekeeping proteins (core compartment), and proteins involved in the developmental stages of T. cruzi. For the first time in trypanosomatids, tRNA sequencing was employed to reveal coadaptation between codon usage and anticodon availability. Despite notable differences in the proteomes of infective and non-infective forms, they exhibited similar pools of tRNAs and similar codon usage preferences. We observed that open chromatin levels of tRNA genes correlate with tRNA expression in non-infective forms, but not in infective forms, suggesting chromatin states do not control the tRNA pool in the latter. Our analysis also revealed a relationship between anticodon:codon pairing modes and protein abundance. Highly expressed mRNAs favored Watson- Crick base pairing, whereas less expressed mRNAs displayed more wobble base pairing. Overall, our findings suggest that protein expression in T. cruzi is influenced by a combination of codon usage bias, tRNA abundance, and anticodon:codon pairing modes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/602108v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1f63f77org.highwire.dtl.DTLVardef@1c0abf8org.highwire.dtl.DTLVardef@5a4988org.highwire.dtl.DTLVardef@1918f49_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Open chromatin analysis in Trypanosoma cruzi life forms highlights critical differences in genomic compartments and developmental regulation at tDNA loci

BackgroundGenomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. ResultsHere, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using genome-wide approaches supported by transmission electron microscopy. The integration of FAIRE and MNase-seq data, two complementary epigenomic approaches, enabled us to identify differences in T. cruzi genome compartments, putative transcriptional start regions and virulence factors. In addition, we also detected developmental chromatin regulation at tRNA loci (tDNA), which seems to be linked to the translation regulatory mechanism required for parasite differentiation. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. ConclusionTaken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

microbiology↗