MRPS28 Human

Q1.1: What is MRPS28 and what is its fundamental role in mitochondrial translation?

MRPS28 (Mitochondrial Ribosomal Protein S28) is a nuclear-encoded component of the small (28S) subunit of the mitochondrial ribosome, essential for translating the 13 mitochondrially-encoded proteins of the oxidative phosphorylation system. Previously known as MRPS35 in some literature, this protein contributes to the unique protein-rich composition of mitoribosomes (approximately 75% protein to 25% rRNA), which contrasts sharply with bacterial ribosomes where this ratio is reversed . This protein-rich nature may compensate for the reduced rRNA content in mitoribosomes and provide additional stabilizing interactions. Methodologically, researchers can study MRPS28's role through ribosome profiling, cryo-electron microscopy, and in vitro translation assays with purified mitochondrial ribosomes.

Q1.2: How has MRPS28 evolved compared to its bacterial counterparts, and what methodological approaches best detect these evolutionary changes?

Evolutionary analysis of MRPS28 requires sophisticated comparative genomics approaches due to high sequence divergence across species . Profile-profile homology searches rather than simple BLAST searches are recommended, as they can detect remote homology even when sequence identity is low. Structure-based alignments considering protein folding patterns (which are often more conserved than primary sequences) and phylogenetic reconstruction using maximum likelihood methods with appropriate substitution models yield the most reliable results. Research has revealed that mammalian MRPS28 shares limited sequence identity with bacterial counterparts, having undergone substantial evolutionary changes. Mitochondrial ribosomes essentially doubled their protein content during eukaryotic evolution, with proteins like MRPS28 either expanding from bacterial ancestors or being recruited from other cellular processes .

Q1.3: What genomic and transcriptomic approaches can accurately characterize MRPS28 gene structure and expression patterns across tissues?

Comprehensive MRPS28 characterization requires integrating multiple technologies:

• Genomic structure analysis: Whole genome sequencing combined with targeted capture sequencing can identify all exons, introns, and regulatory regions of MRPS28 on chromosome 8.

• Expression profiling across tissues: RNA-Seq with sufficient depth (>30 million reads) provides quantitative expression data, with single-cell RNA-Seq offering cellular resolution.

• Transcript isoform identification: Long-read sequencing (Oxford Nanopore or PacBio) can resolve complete transcript structures, including alternative splicing patterns.

• Regulatory element mapping: ChIP-Seq for transcription factors and histone modifications, combined with ATAC-Seq for chromatin accessibility.

These approaches have revealed variable MRPS28 expression across tissues, with particular upregulation in energy-demanding tissues and notably in certain cancer subtypes like Luminal B breast cancer .

Q2.1: How can researchers experimentally determine MRPS28's structural position and interactions within the mitoribosome?

Determining MRPS28's structural context requires complementary approaches:

These approaches have revealed that MRPS28 likely makes multiple contacts with both rRNA and other proteins within the small subunit, contributing to the architecture of the mitochondrial translation machinery .

Q2.2: What are the methodological challenges in studying the functional domains of MRPS28, and how can these be overcome?

Studying MRPS28 functional domains presents several methodological challenges:

• Domain identification: Computational prediction tools may miss eukaryote-specific domains absent in bacterial homologs. Solution: Combine multiple prediction algorithms with evolutionary analysis and experimental validation through truncation studies.

• Mitochondrial import interference: Modifying MRPS28 may affect its mitochondrial targeting. Solution: Carefully design constructs maintaining the N-terminal mitochondrial targeting sequence and verify proper localization using fluorescent tags.

• Assembly disruption: Mutations may prevent incorporation into the mitoribosome. Solution: Use proximity labeling techniques (BioID/TurboID) to assess integration into the ribosomal complex.

• Functional redundancy: Other MRPs may compensate for MRPS28 modifications. Solution: Assess translation of all 13 mtDNA-encoded proteins and conduct ribosome profiling to detect subtle translation defects.

• Tissue-specific effects: Functions may vary across cell types. Solution: Conduct studies in multiple cell lines and tissue-specific models.

Research has suggested MRPS28 has acquired new functional domains absent in bacterial ancestors, potentially contributing to unique aspects of mitochondrial translation regulation .

Q2.3: What biochemical approaches can distinguish MRPS28's role in mitoribosome assembly versus its function in active translation?

Distinguishing between MRPS28's roles in assembly and active translation requires methodically separating these processes:

• Assembly analysis:

  • Sucrose gradient analysis of mitoribosome assembly intermediates in MRPS28-depleted cells

  • Pulse-chase experiments with isotope-labeled MRPs to track assembly kinetics

  • Cryo-EM of assembly intermediates accumulated after MRPS28 depletion

  • Native gel electrophoresis to visualize assembly states

• Translation function analysis:

  • Ribosome footprinting to map translating ribosomes on mitochondrial mRNAs

  • In vitro reconstitution of translation with purified components including wild-type or mutant MRPS28

  • Peptidyl transferase activity assays with assembled mitoribosomes

  • Single-molecule fluorescence microscopy to visualize translation dynamics

• Temporal separation strategies:

  • Rapid MRPS28 depletion using auxin-inducible degron systems after assembly is complete

  • Temperature-sensitive MRPS28 mutants that function in assembly but fail during translation

  • Stage-specific crosslinking to capture interaction partners during assembly versus translation

These approaches allow researchers to determine whether MRPS28 primarily contributes to structural integrity or actively participates in the translation process itself.

Q3.1: How can researchers comprehensively identify and characterize pathogenic MRPS28 mutations?

A systematic approach to identifying and characterizing MRPS28 mutations involves:

• Mutation identification:

  • Whole exome/genome sequencing of patients with suspected mitochondrial translation defects

  • Targeted sequencing of mitochondrial ribosomal protein genes in mitochondrial disease cohorts

  • RNA sequencing to identify splicing defects and expression changes

  • Copy number variation analysis using array CGH or read-depth analysis

• Pathogenicity assessment:

  • Segregation analysis in families

  • Conservation analysis across species

  • Structural modeling to predict impact on protein folding and interactions

  • Functional prediction algorithms (SIFT, PolyPhen, CADD)

• Functional validation:

  • Patient fibroblast studies measuring mitochondrial translation using 35S-methionine labeling

  • Complementation assays in patient cells with wild-type MRPS28

  • CRISPR/Cas9 introduction of mutations in control cells

  • Blue native PAGE to assess OXPHOS complex assembly

  • Respirometry to measure oxygen consumption rates

MRPS28 mutations have been associated with Combined Oxidative Phosphorylation Deficiency 47 (COXPD47) , characterized by impaired mitochondrial translation resulting in multiple OXPHOS defects.

Q3.2: What is the expression profile of MRPS28 across cancer types, and what approaches minimize technical artifacts in expression analysis?

Analyzing MRPS28 expression in cancer requires addressing several technical challenges:

Current research has identified MRPS28 upregulation specifically in Luminal B subtype breast cancer , suggesting potential involvement in the metabolic reprogramming characteristic of this cancer subtype. This subtype-specific expression pattern underscores the importance of analyzing expression with proper stratification of cancer subtypes.

Q3.3: What experimental approaches can determine whether MRPS28 alterations are drivers or passengers in cancer progression?

Determining the functional significance of MRPS28 alterations in cancer requires:

• Causality assessment:

  • CRISPR/Cas9 knockout or knockdown of MRPS28 in cancer cell lines

  • Overexpression of MRPS28 in normal cells to detect oncogenic transformation

  • Xenograft models with MRPS28-modified cells to assess tumor formation capacity

  • Analysis of cancer progression in inducible MRPS28 transgenic animal models

• Mechanism elucidation:

  • Metabolic profiling to detect alterations in energy metabolism pathways

  • Transcriptome analysis to identify downstream effectors

  • ChIP-Seq to map potential transcription factor binding at the MRPS28 locus

  • Protein interaction studies to identify cancer-specific binding partners

• Clinical correlation:

  • Multivariate survival analysis controlling for known prognostic factors

  • Serial sampling during cancer progression to track MRPS28 changes over time

  • Drug sensitivity profiling in cells with different MRPS28 expression levels

  • Analysis of MRPS28 expression in therapy-resistant cancer subpopulations

These approaches would help distinguish whether MRPS28 alterations actively contribute to oncogenesis or merely reflect adaptive responses to altered cellular metabolism in cancer cells.

Q4.1: What is the optimal experimental design for studying MRPS28's role in mitochondrial translation using modern technologies?

A comprehensive experimental design for investigating MRPS28's role in mitochondrial translation:

• Genetic manipulation approaches:

  • CRISPR/Cas9 knockout with rescue constructs containing specific mutations

  • Inducible knockdown using shRNA or degron systems for temporal control

  • Patient-derived cells with naturally occurring MRPS28 mutations

  • Heteroplasmic cybrid cells to assess interaction with mtDNA variants

• Translation assessment methods:

  • Mitochondrial ribosome profiling to capture the translatome at single-nucleotide resolution

  • SILAC pulse labeling combined with mass spectrometry for quantitative proteomics

  • Imaging of nascent peptide synthesis using puromycylation and click chemistry

  • Real-time translation monitoring using reporter constructs with nanoluciferase

• Structural analysis techniques:

  • Cryo-EM of translating mitoribosomes with and without MRPS28 modifications

  • Hydrogen-deuterium exchange mass spectrometry to monitor conformational changes

  • Site-specific crosslinking to capture dynamic interactions during translation

• Data integration:

  • Computational modeling of translation rates and error frequencies

  • Integration of structural, functional, and -omics data into predictive models

  • Correlation of molecular phenotypes with cellular and organismal consequences

This comprehensive approach would provide mechanistic insights into MRPS28's specific contributions to mitochondrial translation beyond its structural role in the ribosome.

Q4.2: How can researchers develop cellular and animal models that accurately recapitulate MRPS28-related mitochondrial diseases?

Developing disease-relevant MRPS28 models requires careful consideration of several factors:

• Cellular models:

  • Patient-derived fibroblasts provide disease-relevant context but may adapt to culture

  • iPSC-derived models allow differentiation into affected tissues (neurons, cardiomyocytes)

  • CRISPR/Cas9 knock-in of specific patient mutations in appropriate cell types

  • Isogenic control generation through correction of mutations in patient cells

  • Consideration of heteroplasmy and mitochondrial DNA background effects

• Animal models:

  • Constitutive knockout may be embryonic lethal; consider conditional approaches

  • Tissue-specific MRPS28 depletion using Cre-lox systems

  • Knock-in of specific patient mutations to recapitulate human disease

  • Drosophila models for high-throughput screening and genetic interaction studies

  • Zebrafish for developmental phenotyping and in vivo drug screening

• Validation criteria:

  • Molecular phenocopying: mitochondrial translation defects, OXPHOS dysfunction

  • Biochemical phenocopying: energetic deficits, metabolite alterations

  • Cellular phenocopying: mitochondrial morphology, calcium handling, stress responses

  • Tissue phenocopying: affected tissue pathology similar to patient biopsies

  • Response phenocopying: similar response to potential therapeutic interventions

The most informative models will combine genetic precision with physiological relevance to the human disease state.

Q4.3: What bioinformatics pipeline can integrate multi-omics data to comprehensively analyze MRPS28 function and interactions?

An integrated bioinformatics pipeline for MRPS28 analysis:

• Data acquisition and processing:

  • Genomic data: WGS/WES for variant calling (GATK, VarScan2)

  • Transcriptomic data: RNA-Seq processed with STAR, Salmon for quantification

  • Proteomic data: MaxQuant for protein identification and quantification

  • Structural data: AlphaFold2 for protein structure prediction

  • Interaction data: STRING, BioGRID, IntAct databases

• Multi-omics integration:

  • Genomic-transcriptomic correlation: eQTL analysis with Matrix eQTL

  • Transcript-protein correlation: RNA-protein abundance correlation analysis

  • Structure-function mapping: Integration of variant impact with structural models

  • Network construction: Weighted gene co-expression network analysis (WGCNA)

• Functional interpretation:

  • Gene set enrichment analysis (GSEA) for pathway analysis

  • Ingenuity Pathway Analysis (IPA) for causal network construction

  • Gene Ontology enrichment with clusterProfiler

  • Disease association analysis using DisGeNET

• Visualization and exploration:

  • Interactive visualization with Cytoscape for networks

  • R Shiny applications for exploration of integrated datasets

  • Circos plots for multi-dimensional data visualization

  • 3D molecular visualization of structural impacts with PyMOL or ChimeraX

This pipeline enables integration of diverse data types to generate comprehensive insights into MRPS28 biology across normal and disease states.

Q5.1: How does MRPS28 contribute to the tissue-specific manifestations of mitochondrial diseases, and what experimental approaches can resolve this question?

Investigating tissue-specific effects of MRPS28 dysfunction requires:

• Comparative tissue analysis:

  • Single-cell RNA-Seq across affected and unaffected tissues in patient samples

  • Proteomics of tissue-specific mitoribosome composition

  • Tissue-specific MRPS28 interactome mapping using BioID/TurboID

  • Isotope tracing to measure tissue-specific metabolic consequences

• Tissue-specific model systems:

  • Development of tissue-specific MRPS28 knockout/knockdown models

  • iPSC differentiation into multiple lineages from the same genetic background

  • Organoid models of affected tissues from patient cells

  • Tissue explant cultures for ex vivo manipulation

• Molecular compensation analysis:

  • Assessment of paralog expression across tissues

  • Identification of tissue-specific translation quality control mechanisms

  • Analysis of nuclear-encoded OXPHOS subunit upregulation as compensation

  • Measurement of mitochondrial biogenesis responses in different tissues

• Vulnerability factor identification:

  • Energy demand profiling across tissues

  • Analysis of tissue-specific mitochondrial dynamics

  • Assessment of antioxidant capacity across tissues

  • Evaluation of tissue-specific mitophagy and mitochondrial quality control

This multi-faceted approach would provide insights into why certain tissues are more vulnerable to MRPS28 dysfunction despite its ubiquitous expression.

Q5.2: What experimental strategies can determine how MRPS28 mutations affect mitoribosome quality control and potentially contribute to proteostatic stress?

Investigating the relationship between MRPS28 and mitochondrial quality control requires:

• Translation fidelity assessment:

  • Reporter systems to measure mistranslation rates

  • Mass spectrometry detection of amino acid misincorporation

  • Ribosome profiling to identify frameshifting or readthrough events

  • In vitro translation assays with purified components to measure error rates

• Protein quality surveillance:

  • Analysis of mitochondrial chaperone recruitment to nascent peptides

  • Ubiquitination profiling of newly synthesized mitochondrial proteins

  • Protein half-life measurements using dynamic SILAC

  • Aggregation propensity analysis of mitochondrial translation products

• Stress response integration:

  • Activation assessment of the mitochondrial unfolded protein response (UPRmt)

  • Analysis of retrograde signaling to the nucleus

  • Integrated stress response (ISR) activation measurement

  • Mitophagy rate quantification in response to translation defects

• Therapeutic targeting opportunities:

  • Small molecule screening for compounds that enhance quality control

  • Testing chaperone-inducing compounds

  • Evaluation of proteasome modulators

  • Assessment of autophagy/mitophagy inducers in disease models

These approaches would reveal how MRPS28 dysfunction might lead to proteostatic stress and identify potential intervention points to mitigate disease phenotypes.

Q5.3: How can researchers investigate potential non-canonical functions of MRPS28 beyond its role in the mitoribosome?

Exploring non-canonical functions of MRPS28 requires innovative approaches:

• Spatial-localization studies:

  • Super-resolution microscopy to detect non-mitoribosomal MRPS28 pools

  • Biochemical fractionation to identify MRPS28 in unexpected compartments

  • Proximity labeling in different cellular compartments

  • Live-cell imaging with tagged MRPS28 under various cellular conditions

• Interactome expansion analysis:

  • Affinity purification-mass spectrometry under different cellular states

  • Yeast two-hybrid screening with full-length and domain-specific constructs

  • Protein complementation assays to validate direct interactions

  • Comparative interactomics between free and ribosome-bound MRPS28

• Functional screening approaches:

  • Transcriptome analysis after acute MRPS28 depletion (before translation effects)

  • Metabolomic profiling focusing on non-mitochondrial pathways

  • Phenotypic screening with domain-specific mutants

  • Synthetic genetic interaction screens to identify non-canonical genetic networks

• Evolutionary analysis:

  • Detection of positively selected regions outside ribosome-binding domains

  • Identification of neo-functionalized domains absent in bacterial ancestors

  • Analysis of tissue-specific isoforms with potentially specialized functions

  • Correlation of domain acquisition with specific eukaryotic functions

This systematic approach would reveal whether MRPS28 has evolved moonlighting functions beyond its primary role in mitochondrial translation, potentially explaining certain disease phenotypes not attributable to translation defects alone.