Recombinant Chicken 28S ribosomal protein S6, mitochondrial (MRPS6)

What is the fundamental function of MRPS6 in mitochondrial translation?

MRPS6 (28S ribosomal protein S6, mitochondrial) is an essential component of the small subunit of mitochondrial ribosomes. It contributes to the structural integrity of the 28S subunit and participates in protein synthesis within the mitochondrion. Mitochondrial ribosomes have a distinct composition from cytoplasmic ribosomes, with approximately 75% protein to 25% rRNA (compared to the reversed ratio in prokaryotic ribosomes) . MRPS6 belongs to the ribosomal protein S6P family and helps translate the 13 proteins encoded by mitochondrial DNA, which are critical components of the oxidative phosphorylation system.

Methodologically, studies of MRPS6 function typically employ techniques such as gene knockout/knockdown, overexpression, and ribosome profiling to assess its contribution to mitochondrial translation efficiency.

How does the structure of MRPS6 differ between avian and mammalian species?

While the search results don't provide specific information about chicken MRPS6 structure, research on mammalian MRPS6 reveals that mitochondrial ribosomal proteins differ significantly in sequence between species, which can make identification by sequence homology challenging . This sequence divergence likely reflects species-specific adaptations in mitochondrial function.

To study structural differences between avian and mammalian MRPS6:

• Perform sequence alignment and phylogenetic analysis of MRPS6 from multiple species

• Use homology modeling based on available structural data

• Express and purify recombinant proteins from different species for comparative structural analysis

• Apply techniques such as X-ray crystallography or cryo-EM to determine high-resolution structures

Despite sequence differences, the core functional domains would likely be conserved to maintain essential ribosomal functions.

What expression patterns does MRPS6 show across different avian tissues?

In avian systems, we would expect higher MRPS6 expression in tissues with high mitochondrial content and metabolic activity, such as:

To study tissue-specific expression patterns, researchers should use quantitative PCR, Western blotting, or immunohistochemistry with chicken-specific primers or antibodies.

What expression systems are most effective for producing recombinant chicken MRPS6?

Based on successful expression of mammalian MRPS6 in research settings, several expression systems could be suitable for recombinant chicken MRPS6:

• Mammalian expression systems: The pCAGGS vector has been successfully used for MRPS6 expression . This approach involves amplification of the MRPS6 gene using specific primers and subcloning into pCAGGS using appropriate restriction sites (EcoRI and NotI were used in the referenced study) .

• Bacterial expression systems: E. coli systems using vectors like pET or pGEX may be suitable for producing moderate amounts of protein, though proper folding of mitochondrial proteins can be challenging.

• Baculovirus-insect cell systems: For higher yields of properly folded protein with appropriate post-translational modifications.

When choosing an expression system, consider:

• The need for post-translational modifications

• Required protein yield

• Downstream applications

• Whether functional studies will be performed

The optimal purification strategy would involve affinity chromatography (if using tagged protein), followed by ion exchange and size exclusion chromatography to achieve high purity.

How can researchers verify the functional activity of recombinant chicken MRPS6?

To confirm that recombinant chicken MRPS6 is functionally active, researchers should employ multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism to verify proper folding

  • Size exclusion chromatography to confirm monomeric state

  • Limited proteolysis to assess domain organization

• Binding studies:

  • Assess binding to other mitochondrial ribosomal components

  • RNA binding assays if MRPS6 interacts directly with ribosomal RNA

• Functional complementation:

  • Express recombinant chicken MRPS6 in cells where endogenous MRPS6 has been knocked down

  • Measure restoration of mitochondrial translation

  • Assess rescue of phenotypes like UPRmt activation or virus resistance

• In vitro translation assays:

  • Reconstitute mitochondrial ribosomes with recombinant MRPS6

  • Measure translation efficiency of reporter constructs

Researchers should look for phenotypic effects similar to those observed with mammalian MRPS6, including modulation of UPRmt markers, changes in reactive oxygen species (ROS) levels, and effects on apoptosis under stress conditions .

What are the critical considerations when designing experiments to study MRPS6 knockdown or overexpression?

When manipulating MRPS6 expression levels in experimental settings, several key considerations should be addressed:

For knockdown experiments:

• Target specificity: Design siRNAs or shRNAs specific to chicken MRPS6 to minimize off-target effects

• Knockdown verification: Confirm reduction at both mRNA (RT-qPCR) and protein (Western blot) levels

• Timing considerations: MRPS6 expression changes dynamically during stress responses, so temporal analysis is crucial

• Cell type selection: Different cell types show varying baseline levels of MRPS6 expression (e.g., higher in Caco2 than HIEC-6 cells in human studies)

For overexpression experiments:

• Expression vector selection: Use vectors with promoters active in avian cells (e.g., pCAGGS with appropriate promoters)

• Expression level control: Excessive overexpression may cause artifacts

• Tag selection: Consider how tags might affect MRPS6 function

• Localization verification: Confirm mitochondrial localization of expressed protein

Experimental readouts to consider:

• UPRmt marker expression (mtHSP70, HSP60, LonP1, ClpP)

• ROS levels using fluorescent probes

• Apoptosis markers (Annexin V, Propidium iodide)

• Mitochondrial translation efficiency

• Response to stressors (high glucose conditions proved informative in mammalian studies)

How does MRPS6 interact with the mitochondrial unfolded protein response (UPRmt)?

Research in mammalian cells has revealed a complex regulatory relationship between MRPS6 and the UPRmt:

• MRPS6 expression is positively regulated by UPRmt activation, but feedback inhibits UPRmt .

• This regulatory circuit involves the transcription factor ATF5:

  • MRPS6 knockdown activates UPRmt in an ATF5-dependent manner

  • MRPS6 overexpression inhibits UPRmt, also in an ATF5-dependent manner

• The feedback mechanism appears crucial for cellular homeostasis:

  • Disruption by MRPS6 knockdown causes UPRmt hyperactivation under high glucose conditions

  • This leads to elevated ROS levels, increased apoptosis, and impaired function

To study this pathway in avian cells, researchers should:

• Analyze expression correlation between MRPS6 and UPRmt markers in chicken cells

• Perform knockdown/overexpression of MRPS6 and measure effects on UPRmt markers

• Investigate the role of chicken ATF5 or equivalent transcription factors

• Examine the effects under normal and stress conditions

Understanding this regulatory network may provide insights into how mitochondrial homeostasis is maintained in avian systems and how it might differ from mammalian systems.

What evidence suggests MRPS6 may function as an antiviral factor?

Compelling evidence from human cell studies indicates that MRPS6 can function as a host restriction factor against viral infection:

• Differential expression of MRPS6 was observed at 48 hours post-infection with porcine deltacoronavirus (PDCoV) in HIEC-6 cells .

• The expression dynamics showed an initial increase followed by a decrease in MRPS6 levels during PDCoV infection .

• Functional studies demonstrated that:

  • Overexpression of MRPS6 significantly inhibited PDCoV infection in HIEC-6 cells

  • Knockdown of MRPS6 in Caco2 cells led to a significant increase in virus titer

• Mechanistically, MRPS6 enhanced the production of IFN-β through interferon pathway activation:

  • MRPS6 had an "augmentative effect" on IFN-β production

  • It promoted production of IRF3, IRF7, STAT1, and STAT2

  • This activation of the interferon pathway impeded viral infection

To investigate whether chicken MRPS6 has similar antiviral properties:

• Test its effect against avian viruses in chicken cell lines

• Examine its impact on avian interferon pathways

• Compare its antiviral efficiency with mammalian MRPS6

• Investigate whether avian viruses have evolved mechanisms to counteract MRPS6

This research direction could yield important insights for avian virology and comparative immunology.

What role does MRPS6 play in cellular metabolism and how is this relevant to metabolic disorders?

Research in mammalian systems has revealed important roles for MRPS6 in metabolic regulation:

• MRPS6 modulates glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells:

  • Knockdown of MRPS6 impairs GSIS

  • Overexpression enhances GSIS in an ATF5-dependent manner

• The metabolic regulatory function of MRPS6 appears mechanistically linked to its role in UPRmt regulation:

  • MRPS6 knockdown causes UPRmt hyperactivation in high glucose conditions

  • This leads to elevated ROS levels and impaired cellular function

  • Conversely, MRPS6 overexpression reduces UPRmt, mitigates high glucose-induced ROS levels and apoptosis

• Genetic evidence supports MRPS6's role in glucose metabolism:

  • SNPs in the MRPS6 gene were associated with 2-hour oral glucose tolerance test results

  • This is an established indicator of impaired glucose tolerance

To study these functions in avian systems, researchers should:

• Investigate MRPS6 expression in chicken pancreatic tissue

• Examine effects of MRPS6 manipulation on glucose metabolism in avian cell models

• Study how MRPS6 levels affect metabolic stress responses

• Explore whether similar genetic variants exist in chicken MRPS6

These studies could provide insights into avian metabolic regulation and potentially inform comparative studies of metabolic disorders between species.

How can researchers optimize protein-protein interaction studies for MRPS6?

Studying MRPS6 protein interactions requires specialized approaches due to its mitochondrial localization:

• Affinity purification techniques:

  • Use a tagged version of MRPS6 (e.g., FLAG, HA, or His)

  • Perform pulldowns under conditions that preserve mitochondrial protein interactions

  • Analyze interacting partners by mass spectrometry

  • Validate key interactions with co-immunoprecipitation

• Proximity labeling approaches:

  • Fuse MRPS6 to BioID or APEX2 enzymes

  • These will biotinylate proteins in close proximity to MRPS6 in living cells

  • Analyze biotinylated proteins to identify the MRPS6 "interactome"

  • This approach is particularly valuable for studying interactions in their native mitochondrial environment

• Crosslinking mass spectrometry:

  • Apply chemical crosslinkers to stabilize transient interactions

  • Identify crosslinked peptides by mass spectrometry

  • This can provide spatial information about interaction interfaces

• Fluorescence-based interaction studies:

  • FRET (Förster Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation)

  • Requires careful design of fusion proteins to maintain mitochondrial targeting

For all approaches, appropriate controls must include:

• Non-mitochondrial proteins to detect non-specific interactions

• Mitochondrial proteins known not to interact with MRPS6

• Validation across multiple cell types and conditions

What approaches can identify the role of MRPS6 in neurodegeneration?

Research has identified MRPS6 as a potential factor in neurodegenerative processes:

• A multiregional gene expression analysis of postmortem brain tissue from Parkinson's disease donors found MRPS6 among 11 genes whose expression was regulated in at least 18 out of 21 brain regions surveyed .

• The consistent alteration of MRPS6 expression across multiple brain regions suggests a potential role in neurodegeneration .

To investigate MRPS6's role in neurodegeneration in avian models:

• Expression analysis:

  • Compare MRPS6 expression in normal versus neurodegenerative avian brain tissues

  • Examine expression patterns across different brain regions

  • Correlate with markers of neurodegeneration

• Functional studies in neuronal cells:

  • Manipulate MRPS6 levels in avian neuronal cultures

  • Assess impact on:

    • Mitochondrial function (membrane potential, ATP production)

    • Oxidative stress and ROS production

    • Neuronal survival under stress conditions

    • UPRmt activation

• In vivo models:

  • Generate avian models with altered MRPS6 expression

  • Examine cognitive and motor function

  • Perform histopathological analysis of brain tissue

• Molecular pathway analysis:

  • Investigate how MRPS6 interacts with known neurodegeneration pathways

  • Examine effects on protein aggregation, a hallmark of many neurodegenerative diseases

  • Study mitochondrial dynamics (fission/fusion) which are often disrupted in neurodegeneration

These approaches could provide valuable insights into the comparative aspects of neurodegeneration across species.

How does MRPS6 coordinate with the interferon pathway in antiviral responses?

Research has revealed that MRPS6 enhances antiviral responses through interaction with the interferon pathway:

• MRPS6 exerts an "augmentative effect" on the production of IFN-β through interferon pathway activation .

• Subsequent investigations demonstrated that MRPS6 promotes the production of key antiviral signaling molecules:

  • IRF3 (Interferon Regulatory Factor 3)

  • IRF7 (Interferon Regulatory Factor 7)

  • STAT1 (Signal Transducer and Activator of Transcription 1)

  • STAT2 (Signal Transducer and Activator of Transcription 2)

• This activation of interferon pathway components impedes viral infection in cellular systems .

To investigate the mechanisms by which MRPS6 coordinates with the interferon pathway:

• Signaling pathway analysis:

  • Examine phosphorylation status of key pathway components (IRF3, STAT1/2)

  • Determine whether MRPS6 directly interacts with pathway components

  • Assess nuclear translocation of transcription factors

• Transcriptional regulation:

  • Perform ChIP-seq to identify whether MRPS6 associates with chromatin

  • Use reporter assays to assess activation of interferon-stimulated response elements (ISREs)

  • Analyze global transcriptional changes following MRPS6 manipulation

• Temporal dynamics:

  • Conduct time-course experiments to determine the sequence of events

  • Assess whether MRPS6's effect is immediate or requires protein synthesis

• Comparative analysis:

  • Compare the interferon pathway activation by MRPS6 across species

  • Determine whether the mechanism is conserved between mammals and birds

Understanding this coordination could reveal novel aspects of mitochondrial-nuclear communication in antiviral immunity.

How can CRISPR-Cas9 technology be optimized for studying MRPS6 function in avian systems?

CRISPR-Cas9 genome editing offers powerful approaches for studying MRPS6 function in avian cells:

• Guide RNA design considerations:

  • Target conserved exons of chicken MRPS6

  • Use chicken-specific genome databases for design

  • Screen multiple gRNAs for efficiency

  • Consider off-target effects using prediction tools

• Delivery methods for avian cells:

  • Lipofection or nucleofection for cultured cells

  • Viral vectors (lentivirus or adeno-associated virus) for harder-to-transfect cells

  • In ovo electroporation for developmental studies

• Editing strategies:

  • Complete knockout: Target early exons to create frameshift mutations

  • Domain-specific mutations: Use homology-directed repair with donor templates

  • Conditional systems: Implement floxed alleles with Cre recombinase

  • Knockin tags: Add reporter genes or epitope tags to study localization and interactions

• Validation approaches:

  • Genomic PCR and sequencing to confirm edits

  • Western blotting to verify protein loss/modification

  • Functional assays to assess mitochondrial translation

  • Phenotypic analysis based on known MRPS6 functions

• Specific applications:

  • Generate isogenic cell lines with and without MRPS6

  • Create cell lines with tagged endogenous MRPS6

  • Introduce human disease-associated variants

  • Study tissue-specific effects using conditional systems

These approaches would enable precise dissection of MRPS6 function in avian systems.

What is the relationship between MRPS6 and the regulation of ROS in mitochondrial homeostasis?

Research in mammalian cells has established important connections between MRPS6 and reactive oxygen species (ROS) regulation:

• MRPS6 knockdown increases ROS levels under high glucose conditions .

• Conversely, MRPS6 overexpression mitigates high glucose-induced ROS levels .

• This regulation appears mechanistically linked to MRPS6's role in UPRmt:

  • Disruption of UPRmt feedback by MRPS6 knockdown causes UPRmt hyperactivation

  • This leads to elevated ROS levels and increased apoptosis

  • The effect is particularly pronounced under metabolic stress (high glucose)

To investigate this relationship in avian systems:

• ROS measurement techniques:

  • Use fluorescent probes like DCFDA to measure cellular ROS

  • Apply mitochondria-specific ROS indicators

  • Measure oxidative damage to proteins, lipids, and DNA

• Mechanistic studies:

  • Determine whether ROS changes are primary or secondary to UPRmt alterations

  • Investigate effects on mitochondrial electron transport chain function

  • Examine antioxidant response pathways (e.g., Nrf2 activation)

• Stress condition analysis:

  • Test multiple stressors beyond glucose (e.g., hypoxia, toxins)

  • Determine cell type specificity of responses

  • Assess acute versus chronic effects

• Intervention studies:

  • Apply antioxidants to determine if they rescue MRPS6 knockdown phenotypes

  • Use mitochondria-targeted antioxidants for specificity

  • Modulate specific ROS sources to identify the primary source

This research direction could reveal important aspects of mitochondrial quality control in avian systems and provide comparative insights with mammalian systems.

How might tissue-specific functions of MRPS6 inform targeted therapeutic approaches?

Understanding tissue-specific roles of MRPS6 could inform therapeutic strategies:

• Tissue expression patterns:

  • Human data shows varying correlation of MRPS6 with UPRmt genes across tissues

  • Particularly strong correlations exist in whole blood, brain, heart, and pancreas

  • Similar analysis in avian tissues would establish comparative expression patterns

• Functional specialization:

  • In pancreatic β-cells, MRPS6 modulates glucose-stimulated insulin secretion

  • In intestinal epithelial cells, MRPS6 exhibits antiviral properties

  • In neuronal tissue, MRPS6 shows altered expression in neurodegeneration

To investigate tissue-specific functions:

• Comparative transcriptomics:

  • Analyze MRPS6 co-expression networks across tissues

  • Identify tissue-specific interaction partners

  • Compare these networks between avian and mammalian systems

• Conditional manipulation approaches:

  • Use tissue-specific promoters for targeted overexpression

  • Apply conditional knockout strategies

  • Compare phenotypic effects across tissues

• Ex vivo tissue studies:

  • Isolate primary cells from different tissues

  • Manipulate MRPS6 expression

  • Compare responses to various stressors

Understanding tissue-specific functions could inform targeted approaches for disorders involving specific tissues, such as metabolic diseases (pancreas), viral infections (epithelial barriers), or neurodegenerative conditions (brain).

What are the most promising future research directions for chicken MRPS6?

Based on current knowledge, several high-priority research directions emerge:

• Evolutionary functional analysis:

  • Compare chicken MRPS6 with mammalian orthologs to identify conserved and divergent functions

  • Investigate whether chicken MRPS6 exhibits the same regulatory relationship with UPRmt

  • Determine if antiviral properties are conserved across species

• Metabolic regulation:

  • Explore the role of chicken MRPS6 in avian glucose metabolism

  • Investigate whether it functions in pancreatic cells similar to mammalian MRPS6

  • Examine its contribution to metabolic adaptations specific to birds (high body temperature, flight)

• Antiviral mechanisms:

  • Test chicken MRPS6 against avian viruses of agricultural importance

  • Determine if it enhances avian interferon pathways

  • Explore potential for enhancing viral resistance in poultry

• Neurodegenerative processes:

  • Investigate MRPS6 expression in avian models of neurodegeneration

  • Determine whether mitochondrial dysfunction in neuronal cells involves MRPS6 dysregulation

  • Compare findings with mammalian studies to identify conserved mechanisms

These research directions could yield valuable insights for comparative biology, avian health, and potentially human disease understanding through evolutionary comparative approaches.