MRPS2 Human

Basic Research Questions

• What is MRPS2 and what is its fundamental role in mitochondrial biology?

  MRPS2 (Mitochondrial Ribosomal Protein S2) is a nuclear-encoded protein that belongs to the ribosomal protein S2 family. It functions as an essential component of the small 28S subunit of mitochondrial ribosomes (mitoribosomes). Mammalian mitochondrial ribosomal proteins are encoded by nuclear genes and help synthesize proteins within the mitochondrion, particularly the 13 proteins encoded by mitochondrial DNA that are critical components of the oxidative phosphorylation (OXPHOS) system .

  Methodologically, researchers can identify MRPS2 through immunoblotting with specific antibodies or through mass spectrometry-based proteomics approaches that detect mitoribosomal components. MRPS2 contains 296 amino acids with a molecular mass of approximately 33 kDa and is part of the mitochondrial small ribosomal subunit (mt-SSU) .

• How does the structure of human mitochondrial ribosomes differ from prokaryotic ribosomes?

  Human mitochondrial ribosomes display several distinctive structural characteristics compared to their prokaryotic counterparts:

  Feature	Mitochondrial Ribosomes	Prokaryotic Ribosomes
  Protein:rRNA ratio	~75% protein to ~25% rRNA	~33% protein to ~67% rRNA
  5S rRNA	Absent	Present
  Structural composition	Small 28S and large 39S subunits	Small 30S and large 50S subunits
  Specialized proteins	Contain unique supernumerary proteins	Lack mitochondria-specific proteins

  These structural differences reflect evolutionary adaptations for mitochondrial-specific functions. Notably, mitochondrial ribosomes have undergone substantial structural remodeling throughout evolution, with a significant loss of ribosomal RNA while acquiring unique protein subunits located on the periphery of the ribosomal structure .

• What is the genomic organization and expression pattern of MRPS2 in humans?

  The MRPS2 gene is located on chromosome 9 (position 9:138395678-138395821 for a specific amplicon) and encodes the mitochondrial ribosomal protein S2. The gene has multiple exons and can undergo alternative splicing, producing different transcript variants . It has several synonyms including MRP-S2, S2mt, and CGI-91.

  MRPS2 has a standard gene structure with exonic regions that can be targeted for PCR amplification (as shown in validation studies with an amplicon length of 114 bp) . Expression studies indicate that MRPS2 is ubiquitously expressed across tissues, which is consistent with the fundamental role of mitochondria in cellular energy production. The Allen Brain Atlas data suggests differential expression across brain regions, highlighting potential tissue-specific regulation of this gene .

Advanced Research Questions

• What pathogenic mutations have been identified in MRPS2 and what are their phenotypic consequences?

  Bi-allelic mutations in MRPS2 cause a mitochondrial disease designated as Combined Oxidative Phosphorylation Deficiency 36 (COXPD36). The clinical presentation includes:

  • Sensorineural hearing impairment

  • Mild developmental delay

  • Hypoglycemia

  • Lactic acidemia

  • Combined OXPHOS deficiencies

  Recent findings have expanded the phenotypic spectrum to include:

  • Microcephaly

  • Joint hypermobility

  • Autistic features

  Experimental studies have shown that pathogenic MRPS2 variants destabilize the protein, thereby impairing mt-SSU assembly. This leads to inhibition of mitochondrial translation and multiple OXPHOS deficiencies. Reintroduction of wild-type MRPS2 restores mitochondrial translation and OXPHOS assembly in patient fibroblasts, confirming the causal relationship between MRPS2 deficiency and disease .

• How do MRPS2 mutations affect mitoribosome assembly and function at the molecular level?

  Pathogenic MRPS2 mutations impair mitoribosome assembly through several mechanisms:

  1. Reduction in steady-state amounts of mutant MRPS2 protein

  2. Destabilization of the small mitoribosomal subunit (28S)

  3. Prevention of complete mitoribosome assembly

  Complexome profiling of fibroblasts from affected individuals reveals specific assembly defects of the small mitoribosomal subunit. The impaired assembly inhibits mitochondrial translation, resulting in combined OXPHOS deficiency detectable in patients' muscle and liver biopsies as well as in cultured skin fibroblasts .

  Functionally, this translates to:

  • Decreased synthesis of mitochondrially-encoded OXPHOS components

  • Reduced activity of respiratory chain complexes I, III, IV, and V

  • Impaired ATP production via oxidative phosphorylation

  • Metabolic consequences including lactic acidosis and hypoglycemia

• How does the mitochondrial small ribosomal subunit (mt-SSU) assembly process occur and what role does MRPS2 play in this pathway?

  The assembly of the mitochondrial small ribosomal subunit follows a modular process involving multiple factors. Research using CRISPR-mediated knockouts of all 14 supernumerary mitochondrial ribosomal proteins of the small subunit has revealed that each knockout leads to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis .

  MRPS2 appears to be essential for proper mt-SSU assembly. In the assembly pathway:

  1. Initial formation of core mt-SSU components occurs

  2. MRPS2 incorporation is required for stable assembly progression

  3. Absence of MRPS2 prevents completion of the mt-SSU

  4. Complete mt-SSU is necessary for association with the large subunit to form functional mitoribosomes

  Interestingly, research has shown that knockouts of individual supernumerary proteins affect the stability of mS37 (MRPS37/CHCHD1), suggesting this protein may act as a regulatory checkpoint in mitoribosome assembly. A redox-regulated CX9C motif in mS37 is essential for protein stability, indicating a potential mechanism to regulate mitochondrial protein synthesis in response to cellular redox state .

• What methodological approaches are most effective for studying MRPS2 function in experimental systems?

  Several complementary approaches are effective for investigating MRPS2 function:

  Technique	Application	Advantage
  CRISPR-Cas9 genome editing	Generate knockout cell lines	Allows complete elimination of protein function
  Patient-derived fibroblasts	Study disease-causing mutations	Provides physiologically relevant context
  Complementation studies	Functional rescue experiments	Confirms causality of identified mutations
  Complexome profiling	Analyze mitoribosome assembly	Reveals composition of assembly intermediates
  Metabolic labeling	Measure mitochondrial translation	Directly assesses functional impact on protein synthesis
  Blue Native PAGE	Assess OXPHOS complex assembly	Evaluates downstream effects on respiratory chain
  Cryo-EM	Structural analysis of mitoribosomes	Provides high-resolution structural insights

  When using these approaches, researchers should supplement culture media with uridine and pyruvate to bypass any potential growth defects caused by respiratory chain deficiency . This methodological consideration is crucial for maintaining viable cells with severe mitoribosomal defects.

• How do MRPS2 defects contribute to tissue-specific disease manifestations despite its ubiquitous expression?

  The tissue-specific manifestations of MRPS2 deficiency (hearing loss, hypoglycemia, neurological symptoms) despite its ubiquitous expression can be explained by several factors:

  1. Different tissues have varying energy demands and dependence on mitochondrial function

  2. Evidence suggests tissue-specific mitoribosomal protein profiles exist

  3. Recent research indicates that specific mitoribosomal proteins might regulate the translation of specific respiratory chain subunits

  4. MRPS2 mutations can result in isolated complex IV deficiency in muscle, rather than universal dysfunction of all respiratory chain enzymes

  This differential impact suggests specialized regulation of specific subunit translation in different tissues. The observation that defects in the translation machinery can result in isolated complex IV deficiency in muscle provides an explanation for the different mitochondrial ribosomal profiles among tissues, and consequently the tissue-specific manifestations of MRP-related diseases .

Technical and Analytical Questions

• What are the optimal diagnostic approaches for detecting MRPS2 deficiency in clinical samples?

  A comprehensive diagnostic approach for MRPS2 deficiency should include multiple techniques:

  1. Genetic testing: Next-generation sequencing (exome or genome sequencing) to identify variants in MRPS2. Deep intronic variants may be missed by exome sequencing, necessitating genome sequencing .

  2. Transcript analysis: RNA sequencing to detect abnormal splicing patterns, expression levels, or deep intronic variants that create cryptic splice sites .

  3. Protein analysis:

     • Immunoblotting to assess MRPS2 protein levels in patient fibroblasts or tissue biopsies

     • Blue Native PAGE to evaluate OXPHOS complex assembly

     • Complexome profiling to analyze mitoribosome assembly defects

  4. Functional studies:

     • Measurement of respiratory chain complex activities in affected tissues

     • Assessment of mitochondrial translation rates using metabolic labeling

     • Oxygen consumption measurements to evaluate mitochondrial respiration

  5. Relative Complex Abundance analysis: A proteomics-based method that can identify defects in OXPHOS disorders with high sensitivity, potentially useful for functional validation or prioritization in rare diseases where protein complex assembly is disrupted .

  The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect .

• How can researchers distinguish between different mitoribosomal protein defects that present with similar clinical and biochemical phenotypes?

  Distinguishing between different mitoribosomal protein defects requires a multi-faceted approach:

  1. Comprehensive genetic analysis:

     • Targeted sequencing of all known mitoribosomal protein genes

     • Whole exome or genome sequencing with specific analysis of mitoribosome-related genes

     • RNA sequencing to detect splicing abnormalities or expression changes

  2. Protein-level investigations:

     • Immunoblotting for specific mitoribosomal proteins to identify which components are reduced

     • Complexome profiling to determine the specific stage at which mitoribosome assembly is disrupted

     • Mass spectrometry-based proteomics to assess the relative abundance of all mitoribosomal proteins

  3. Structural analysis:

     • Cryo-EM of purified mitoribosomal complexes to identify structural abnormalities

     • Analysis of mitoribosome assembly intermediates that accumulate in patient cells

  4. Clinical correlation:

     • Certain mitoribosomal protein defects have characteristic clinical features (e.g., defects in MRPS16, MRPS22, and MRPS25 are associated with corpus callosum abnormalities)

     • MRPS2 deficiency specifically presents with sensorineural hearing loss, hypoglycemia, and developmental delay

  These approaches should be used in combination to achieve accurate diagnosis and differentiation between the various mitoribosomal protein defects.

• What challenges exist in studying mitoribosome assembly and how can they be overcome?

  Studying mitoribosome assembly presents several technical challenges:

  Challenge	Solution	Methodological Considerations
  Dynamic assembly process	Capture assembly intermediates	Deplete or overexpress assembly factors like GTPBP5
  Large complex size	High-resolution structural techniques	Use cryo-EM for intact complex visualization
  Multiple assembly factors	Systematic analysis approach	CRISPR-based knockout of individual components
  Growth defects in deficient cells	Media supplementation	Add uridine and pyruvate to bypass respiratory deficiency
  Complex structure-function relationships	Combined structural and functional approaches	Integrate cryo-EM with functional assays
  Discriminating assembly factors from structural components	Careful experimental design	Use complementation studies and structural analysis

  The study by Saveanu et al. (2021) provides an excellent methodological example by determining cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factors. This approach captured consecutive steps during mitoribosomal large subunit biogenesis and revealed the coordinated action of nine assembly factors in the final steps of 16S rRNA folding, methylation, and peptidyl transferase center completion .

• How might insights from MRPS2 research inform therapeutic strategies for mitochondrial translation disorders?

  Research on MRPS2 and other mitoribosomal proteins provides several potential therapeutic avenues:

  1. Gene therapy approaches:

     • Delivery of functional MRPS2 gene to affected tissues

     • Targeted gene editing to correct pathogenic mutations

     • Research has shown that reintroduction of wild-type MRPS2 restores mitochondrial translation and OXPHOS assembly in patient fibroblasts

  2. Small molecule interventions:

     • Compounds that stabilize partially assembled mitoribosomes

     • Molecules that promote read-through of premature termination codons

     • Modulators of mitochondrial translation efficiency

  3. Metabolic bypasses:

     • Strategies to increase glycolytic ATP production

     • Supplements that can provide alternative energy substrates

     • Approaches to enhance mitochondrial biogenesis

  4. RNA therapeutics:

     • Antisense oligonucleotides to correct splicing defects

     • RNA editing technologies to correct point mutations

     • mRNA delivery to provide functional protein

  5. Precision mitochondrial medicine:

     • Patient-specific interventions based on the exact molecular defect

     • Tailored treatment approaches considering tissue-specific manifestations

     • Biomarker-guided therapy to monitor treatment efficacy

  Experimental models using CRISPR-engineered cell lines and patient-derived cells provide valuable platforms for screening potential therapeutic compounds and approaches before moving to more complex in vivo models or clinical trials .