Biology subjects

Rodriguez-Mias, R. A.

Publications and source records attributed to Rodriguez-Mias, R. A..

12 recordsLinked to original sources

bioRxiv2025

Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae

Transfer RNAs (tRNAs) ensure accurate decoding of the genetic code. However, mutations in tRNAs can lead to mis-incorporation of an amino acid that differs from the genetic message in a process known as mistranslation. As mistranslating tRNAs modify how the genetic message is decoded, they have potential as therapeutic tools for diseases caused by nonsense and missense mutations. Despite this, they also produce proteome-wide mis-made proteins which can disrupt proteostasis. To better understand the impact of mistranslating tRNA variants, we profile the proteome and phosphoproteome of yeast expressing three different mistranslating tRNAs. While the overall impacts were similar, the extent of growth defects and proteome changes varied with the substitution type. Although the global impacts were modest, mistranslation influenced key cellular processes, including proteostasis, cell cycle and translation. These findings highlight the need to consider cellular consequences when developing mistranslating tRNAs for therapeutic applications.

systems biology↗

bioRxiv2025

R2HaPpY: Rapid-robust phosphotyrosine peptide enrichment using HaloTag-Src SH2 pY superbinder

Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, systems-level analysis of phosphotyrosine signaling remains a challenge due to costly enrichment reagents and labor-intensive protocols. We previously established an automated phosphotyrosine enrichment method for preparing 96 samples in parallel. Here, we further optimize this method by fusing an SH2 phosphotyrosine superbinder to the HaloTag protein. This allows simple and cost-effective preparation of enrichment beads directly from bacterial lysate, expediting reagent preparation from days to hours. Additionally, our new reagent binds phosphotyrosine peptides at higher efficiency than other enrichment reagents. Using this reagent, we detect and quantify 1,651 unique phosphotyrosine sites from EGF stimulated HeLa cells using only [~]1 mg of input peptides per replicate. These include 878 regulated pY sites, many of which are low abundance and not previously detected or annotated as EGF-responsive. This streamlined and sensitive method facilitates comprehensive, quantitative mapping of tyrosine phosphorylation dynamics, enabling broader integration of phosphotyrosine signaling into multiomic and network-level models across diverse biological systems and disease states.

systems biology↗

bioRxiv2024

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

molecular biology↗

bioRxiv2024

Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment

To bridge the gap between bench and bedside, there is a need for more faithful models of human cancers that can recapitulate key features of the human tumor microenvironment (TME) and simultaneously facilitate large-scale drug tests. Our recently developed microdissection method optimizes the yield of large numbers of cuboidal microtissues (''cuboids '', ~(400 m)3 ) from a tumor biopsy. Here we demonstrate that cuboids from syngeneic mouse tumor models and human tumor biopsies retain a complex TME, making them amenable for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as cellular architecture, cytokine secretion, proteomics profiles, and response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong cytokine expression and drug responses, including to immunotherapy. Overall, our results suggest that cuboids could provide essential therapeutic information for personalized oncology applications and could help the development of TME-dependent therapeutics and cancer disease models, including for clinical trials.

cancer biology↗

bioRxiv2023

Multidimensional proteomics identifies molecular trajectories of cellular aging and rejuvenation

The declining capacity of cells to maintain a functional proteome is a major driver of cellular dysfunction and decreased fitness in aging. Here we assess the impact of aging on multiple proteome dimensions, which are reflective of function, across the replicative lifespan of Saccharomyces cerevisiae. We quantified protein abundance, protein turnover, protein thermal stability, and protein phosphorylation in mother yeast cells and their derived progeny at different ages. We find progressive and cumulative proteomic alterations that are reflective of dysregulation of complex assemblies, mitochondrial remodeling, post-translational activation of the AMPK/Snf1 energy sensor in mother cells, and an overall shift from biosynthetic to energy-metabolic processes. Our multidimensional proteomic study systematically corroborates previous findings of asymmetric segregation and daughter cell rejuvenation, and extends these concepts to protein complexes, protein phosphorylation, and activation of signaling pathways. Lastly, profiling age-dependent proteome changes in a caloric restriction model of yeast provided mechanistic insights into longevity, revealing minimal remodeling of energy-metabolic pathways, improved mitochondrial maintenance, ameliorated protein biogenesis, and decreased stress responses. Taken together, our study provides thousands of age-dependent molecular events that can be used to gain a holistic understanding of mechanisms of aging.

systems biology↗

bioRxiv2022

Probing the genetic code and impacts of mistranslation using tRNAAla anticodon variants

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single-or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C rich anticodons resulted in larger growth deficits than A/U rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

molecular biology↗

bioRxiv2022

The regulatory landscape of the yeast phosphoproteome

The cellular ability to react to environmental fluctuations depends on signaling networks that are controlled by the dynamic activities of kinases and phosphatases. To gain insight into these stress-responsive phosphorylation networks, we generated a quantitative mass spectrometry-based atlas of early phosphoproteomic responses in Saccharomyces cerevisiae exposed to 101 environmental and chemical perturbations. We report phosphosites on 59% of the yeast proteome, with 18% of the proteome harboring a phosphosite that is regulated within 5 minutes of stress exposure. We identify shared and perturbation-specific stress response programs, uncover dephosphorylation as an integral early event, and dissect the interconnected regulatory landscape of kinase-substrate networks, as we exemplify with TOR signaling. We further reveal functional organization principles of the stress-responsive phosphoproteome based on phosphorylation site motifs, kinase activities, subcellular localizations, shared functions, and pathway intersections. This information-rich map of 25,000 regulated phosphosites advances our understanding of signaling networks. HighlightsO_LIUltra-deep reference yeast phosphoproteome covers 36,000 phosphorylation sites and reveals general principles of eukaryotic protein phosphorylation. C_LIO_LIHigh-dimensional quantitative atlas of early phosphoproteomic responses of yeast across 101 environmental and chemical perturbations identifies 25,000 regulated perturbation-phosphosite pairs. C_LIO_LIIdentification of shared and perturbation-specific stress response phosphorylation programs reveals the importance of dephosphorylation as an early stress response. C_LIO_LIDissection of the TOR signaling network uncovers subnetworks with differential stress responsiveness and points of pathway cross-talk C_LIO_LIIdentification of functional organization of the phosphoproteome by dimensionality reduction and co-regulation analysis. C_LI

systems biology↗

bioRxiv2022

Coisolation of peptide pairs for peptide identification and MS/MS-based quantification

SILAC-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a data dependent acquisition strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. In an initial feasibility experiment, this peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions, which are difficult to deconvolute in MS1-based quantification. This study demonstrates the initial feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

systems biology↗

bioRxiv2022

Proteome-wide identification of amino acid substitutions deleterious for protein function

DNA sequencing has led to the discovery of millions of mutations that change the encoded protein sequences, but the impact of nearly all of these mutations on protein function is unknown. We addressed this scarcity of functional data by developing Miro, a proteomic technology that uses mistranslation to introduce amino acid substitutions and biochemical assays to quantify functional differences of thousands of protein variants by mass spectrometry. We apply this technology to the proteome of yeast to reveal amino acid substitutions that impact protein structure, ligand binding, protein-protein interactions, protein post-translational modifications, and protein thermal stability. Adapting Miro to human cells will provide a means to efficiently accelerate our mechanistic interpretation of genomic mutations to predict disease risk.

genomics↗

bioRxiv2021

DIAmeter: Matching peptides to data-independent acquisition mass spectrometry data

Tandem mass spectrometry data acquired using data independent acquisition (DIA) is challenging to interpret because the data exhibits complex structure along both the mass-to-charge (m/z) and time axes. The most common approach to analyzing this type of data makes use of a library of previously observed DIA data patterns (a "spectral library"), but this approach is expensive because the libraries do not typically generalize well across laboratories. Here we propose DIAmeter, a search engine that detects peptides in DIA data using only a peptide sequence database. Unlike other library-free DIA analysis methods, DIAmeter supports data generated using both wide and narrow isolation windows, can readily detect peptides containing post-translational modifications, can analyze data from a variety of instrument platforms, and is capable of detecting peptides even in the absence of detectable signal in the survey (MS1) scan.

bioinformatics↗

bioRxiv2020

Identification of phosphosites that alter protein thermal stability

Proteomics has enabled the cataloguing of 100,000s of protein phosphorylation sites 1, however we lack methods to systematically annotate their function. Phosphorylation has numerous biological functions, yet biochemically all involve changes in protein structure and interactions. These biochemical changes can be recapitulated by measuring the difference in stability between the protein and the phosphoprotein. Building on recent work, we present a method to infer phosphosite functionality by reliably measuring such differences at the proteomic scale.

systems biology↗

bioRxiv2019

Automated high-throughput proteome and phosphoproteome analysis using paramagnetic bead technology

Recent developments in proteomics have enabled signaling studies where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is robust, versatile, high-throughput, and achieves higher sensitivity than classical protocols. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.

systems biology↗