RPLP1 Human

What is RPLP1 and what is its fundamental role in human biology?

RPLP1 (Ribosomal Protein Large P1) is a component of the 60S subunit of the ribosome that forms part of the ribosomal stalk structure. While primarily involved in protein synthesis, research demonstrates that RPLP1 exerts "translation fine-tuning" rather than affecting global protein synthesis. This selective influence impacts specific protein subsets involved in protein folding, unfolded protein response, cell death, protein transport, and signal transduction . RPLP1 is crucial for embryonic development, particularly in the nervous system, and plays significant roles in cell proliferation and survival mechanisms.

How does RPLP1 expression vary across human tissues and development?

RPLP1 shows distinct expression patterns across human tissues as documented in the Human Protein Atlas, which provides comprehensive expression data from 44 normal tissue types at both mRNA and protein levels . In the brain, RPLP1 expression has been characterized across various regions in human, mouse, and pig brains . RPLP1 appears particularly crucial during embryonic development, as evidenced by studies showing that RPLP1 heterozygosity in mice leads to body size reductions, male infertility, systemic abnormalities, and high frequency of early postnatal death .

What cellular processes are most dependent on proper RPLP1 function?

Cell proliferation and survival are highly dependent on RPLP1 function. Experimental studies have demonstrated that RPLP1 deletion leads to proliferation arrest and premature senescence in primary mouse embryonic fibroblasts (pMEFs) . In the developing neocortex, RPLP1 knockout causes progenitor cell proliferation arrest and apoptosis due to dysregulation of key cell cycle and apoptosis regulators, including cyclin A, cyclin E, p21^CIP1, p27^KIP1, and p53 . Additionally, RPLP1 influences protein expression patterns related to protein folding and the unfolded protein response without affecting global protein synthesis rates .

What are validated methods for detecting RPLP1 expression in human tissues?

For protein detection, immunohistochemistry (IHC) using Anti-RPLP1 antibody (Abcam 121190) at a 1:300 dilution has been validated with the VectaStain ABC system . For semi-quantitative assessment of RPLP1 protein expression, the H-Score system has been effectively implemented to quantify expression in different cell types within regions of interest, scoring signal intensity as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong) and calculating the percentage of cells at each intensity level .

For mRNA quantification, qRT-PCR using the following validated primers has been documented:

RPLP1 V1:

• Forward: 5′-TGACAGTCACGGAGGATAAGA-3′

• Reverse: 5′-CCAGGCCAAAAAGGCTCAAC-3′

RPLP1 V2:

• Forward: 5'-CTCACTTCATCCGGCGACTA-3'

• Reverse: 5′-GCCAGGGCCGTGACTGT-3′

The 2-delta-delta CT method with human 18S primers for normalization has been used for calculating fold-change values in expression studies .

What cell culture models are appropriate for studying RPLP1 function?

The 12Z human endometriotic cell line has been successfully used to study RPLP1 function through shRNA knockdown experiments . The methodology involves:

• Plating cells at 1×10^5 cells/well in six-well plates in DMEM:F12 with Pen-Strep and 5% FBS for 24h

• Transducing cells with lentiviral particles expressing either RPLP1-targeting shRNA or non-targeting shRNA controls

• Maintaining cells in media with puromycin (2 μg/mL) to select positively infected cells

• Confirming viral infection through eGFP protein expression

• Assessing cell survival through Trypan blue exclusion at 96h post-puromycin treatment

Primary mouse embryonic fibroblasts (pMEFs) have also been established as valuable models, as RPLP1 deletion in these cells leads to proliferation arrest and premature senescence, allowing for investigation of RPLP1's role in cell cycle regulation .

How can animal models advance understanding of RPLP1 function in humans?

Mouse models with germline disruption of RPLP1 and conditional knockout in the central nervous system (RPLP1 CNS∆) have provided critical insights into RPLP1 function that may translate to human biology . Key experimental approaches include:

• Generating heterozygous RPLP1 knockout mice to study systemic effects, which revealed body size reductions, male infertility, various tissue abnormalities, and high frequency of early postnatal death

• Creating brain-specific knockouts (RPLP1 CNS∆) to investigate neurological functions, which demonstrated perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain, and ganglionic eminence

• Utilizing experimentally induced endometriosis mouse models to study RPLP1 expression in ectopic tissues, which showed that ectopic lesion tissue expressed robust levels of RPLP1 protein predominantly in the epithelium, similar to patterns observed in human lesions

• Correlating RPLP1 expression with physiological outcomes, such as the association between endometriosis-related RPLP1 expression and increased visceromotor response to vaginal balloon distension (a measure of pain)

What is the evidence for RPLP1's role in endometriosis pathogenesis?

Multiple lines of evidence support RPLP1's involvement in endometriosis:

• Differential expression: RPLP1 mRNA and protein are significantly higher in ectopic lesion tissue compared to paired eutopic endometrium, with immunohistochemical localization revealing predominant expression in epithelial cells

• Functional impact: Stable knockdown of RPLP1 protein in endometriotic cells results in a significant decrease in cell survival in vitro, suggesting RPLP1 promotes lesion survival

• Animal model validation: Mouse models of experimentally induced endometriosis recapitulate the pattern of elevated RPLP1 expression in ectopic lesions, primarily in epithelial cells

• Clinical correlation: In mouse models, RPLP1 expression in endometriotic lesions is associated with increased vaginal sensitivity, a validated measure of endometriosis-induced pain

These findings collectively support that RPLP1 contributes to endometriosis pathophysiology through promoting cell proliferation and/or survival in ectopic endometrial tissue.

How does RPLP1 dysfunction impact neurodevelopment?

RPLP1 is crucial for proper neurodevelopment as demonstrated in mouse models:

• CNS-specific RPLP1 knockout (RPLP1 CNS∆) in mice resulted in perinatal lethality and significant brain atrophy, with size reductions specifically in the neocortex, midbrain, and ganglionic eminence

• At the cellular level, RPLP1 knockout in the neocortex caused progenitor cell proliferation arrest and apoptosis

• Molecular analysis revealed that this neuronal phenotype was due to dysregulation of key cell cycle and apoptosis regulators, including cyclin A, cyclin E, p21^CIP1, p27^KIP1, and p53

• Rather than affecting global protein synthesis, RPLP1 deletion altered the expression patterns of specific protein subsets involved in protein folding, unfolded protein response, cell death, protein transport, and signal transduction

These findings suggest that RPLP1-mediated translation "fine-tuning" is essential for proper brain development, particularly for neural progenitor proliferation and survival.

What is the relationship between RPLP1 and cancer?

The available research indicates connections between RPLP1 and cancer:

• RPLP1 overexpression has been associated with tumorigenesis, suggesting potential oncogenic properties

• Given RPLP1's established role in promoting cell proliferation and survival, its dysregulation could contribute to abnormal cell growth characteristic of cancer

• The regulatory effect of RPLP1 on cell cycle proteins (cyclins A and E) and tumor suppressors (p21, p27, p53) provides a molecular mechanism by which RPLP1 could influence cancer development or progression

• RPLP1's selective regulation of protein subsets involved in cell death and signal transduction pathways suggests it may influence cancer-related cellular processes beyond simple proliferation

The Human Protein Atlas includes disease-related keywords for RPLP1 that incorporate cancer-related genes and FDA-approved drug targets, indicating further associations with cancer that warrant investigation .

How does RPLP1 achieve "translation fine-tuning" without affecting global protein synthesis?

RPLP1 deletion studies revealed a surprising selectivity in translational regulation. While global protein synthesis remained unchanged in RPLP1-deleted cells, expression patterns of specific protein subsets were altered . This selective regulation suggests several possible mechanisms:

• RPLP1 may preferentially affect ribosomes translating specific mRNA populations, potentially through recognition of particular RNA structures or sequences

• As part of the ribosomal stalk, RPLP1 might selectively recruit specific translation factors that preferentially regulate certain mRNAs

• RPLP1 could be involved in forming specialized ribosomes with altered translational preferences or efficiencies for specific mRNA subsets

• The selective effect might result from RPLP1's influence on mRNA-specific translation factors rather than core ribosomal functions

How might integrated transcriptomic approaches advance understanding of RPLP1 function?

Advanced transcriptomic approaches can provide new insights into RPLP1 function:

• Combined spatial and single-cell approaches: Integration of single-nucleus RNA-sequencing (snRNA-seq) with spatially-resolved transcriptomics (SRT) can map RPLP1 expression with both cellular resolution and spatial context

• Non-negative matrix factorization (NMF): This computational approach can define gene expression patterns within snRNA-seq data and infer their expression in SRT data, potentially revealing RPLP1 co-expression networks and spatial domains

• Cross-species translation: NMF can be used to translate information from animal models into human brain datasets to make predictions across species about functional properties of RPLP1-expressing cells

• Inference of functional roles: By integrating human expression data with rodent datasets that include information on circuit connectivity and neural activity, researchers can make predictions about potential functions of RPLP1 in spatially-defined cellular populations

These approaches are particularly valuable for studying RPLP1 in complex tissues like the brain, where both cellular identity and spatial organization are critical for understanding function.

What are the molecular mechanisms by which RPLP1 regulates cell proliferation and apoptosis?

Research reveals several potential mechanisms through which RPLP1 regulates cell proliferation and apoptosis:

• Cell cycle regulator expression: RPLP1 knockout affects expression of key cell cycle proteins including cyclin A and cyclin E, which are essential for cell cycle progression

• CDK inhibitor regulation: RPLP1 influences levels of p21^CIP1 and p27^KIP1, critical negative regulators of cell cycle progression

• p53 pathway modulation: RPLP1 affects p53 expression, a master regulator of both cell cycle arrest and apoptosis

• Selective translational control: Rather than affecting all proteins equally, RPLP1 selectively influences translation of specific subsets of mRNAs involved in protein folding, unfolded protein response, cell death, and signal transduction

• Cell-type specific effects: In endometriotic epithelial cells, RPLP1 appears to be particularly critical for survival, as its knockdown significantly decreases cell viability

The convergence of these mechanisms explains RPLP1's profound influence on cell proliferation, with its absence leading to proliferation arrest and premature senescence in fibroblasts and progenitor cell proliferation arrest and apoptosis in the developing neocortex .

What experimental design considerations are critical when investigating tissue-specific RPLP1 functions?

When investigating tissue-specific functions of RPLP1, researchers should consider:

• Tissue heterogeneity: Given RPLP1's predominant localization to epithelial cells in some contexts , single-cell approaches may be necessary to resolve cell type-specific functions

• Temporal dynamics: RPLP1's critical role in development suggests developmental timing is important; experimental designs should account for potential stage-specific functions

• Conditional knockout approaches: Brain-specific knockouts revealed functions not evident in heterozygous models , suggesting tissue-specific deletion is valuable for uncovering specialized roles

• Systematic sampling: When examining structures with diverse subregions (like hippocampus), inconsistent sampling can lead to cellular composition differences between donors, potentially confounding results

• Integration of spatial context: Combining cellular resolution techniques (snRNA-seq) with spatial methods (SRT) provides complementary insights that neither approach alone can offer

• Cross-species validation: Translating findings between human samples and animal models requires careful consideration of species differences in expression patterns and function

How can researchers distinguish between RPLP1's canonical ribosomal functions and potential extraribosomal roles?

Distinguishing between RPLP1's ribosomal and potential extraribosomal functions requires specialized experimental approaches:

• Domain-specific mutations: Creating mutations that specifically disrupt RPLP1's incorporation into ribosomes versus other potential interactions

• Temporal separation: Using rapid induction/repression systems to distinguish immediate translation effects from secondary consequences

• Polysome profiling: Examining whether RPLP1 manipulations alter global polysome profiles or specific mRNA translation patterns

• Protein-protein interaction studies: Identifying RPLP1 interaction partners outside the ribosome through techniques like proximity labeling or co-immunoprecipitation

• Subcellular localization analysis: Determining whether RPLP1 localizes to non-ribosomal compartments under specific conditions

• Selective mutant complementation: Testing whether specific RPLP1 mutants can rescue translation defects but not other phenotypes (or vice versa)

The finding that RPLP1 deletion affects specific protein subsets rather than global translation suggests specialized ribosomal functions that could be mechanistically separated from canonical roles.

What analytical frameworks best capture RPLP1's influence on selective protein synthesis?

To understand RPLP1's selective influence on protein synthesis, these analytical approaches are recommended:

These complementary approaches can together elucidate the mechanisms by which RPLP1 exerts its "fine-tuning" effect on translation of specific protein subsets .

What are the most promising therapeutic applications targeting RPLP1?

Based on current understanding of RPLP1 function, several therapeutic directions warrant investigation:

• Endometriosis treatment: Given RPLP1's elevated expression in endometriotic lesions and its role in lesion cell survival , targeted inhibition might provide a novel treatment approach

• Cancer therapeutics: RPLP1's association with tumorigenesis and its influence on cell proliferation pathways suggest it could be a target for anti-cancer strategies, particularly in cancers showing RPLP1 overexpression

• Neurodevelopmental disorders: Understanding RPLP1's critical role in brain development could inform approaches for treating or preventing certain neurodevelopmental conditions

• Selective translation modulation: RPLP1's ability to regulate specific protein subsets without disrupting global translation represents a potentially powerful mechanism for selective therapeutic intervention

• Biomarker development: RPLP1 expression patterns could serve as diagnostic or prognostic biomarkers for conditions including endometriosis or potential cancers

Each of these directions requires further research to validate RPLP1 as an effective therapeutic target and develop specific intervention strategies.

What technological advances would most benefit RPLP1 research?

Several emerging technologies would significantly advance RPLP1 research:

• Improved spatial transcriptomics with single-cell resolution: Current SRT methods could be enhanced to provide true single-cell resolution while maintaining spatial context

• In vivo translational dynamics imaging: Technologies for visualizing translation of specific mRNAs in living cells and tissues would clarify RPLP1's dynamic regulatory effects

• Ribosome-specific proximity labeling: Methods to identify proteins specifically associated with RPLP1-containing ribosomes would reveal potential mechanistic partners

• Single-molecule translation imaging: Techniques for visualizing individual ribosomes during translation would help understand how RPLP1 influences translation kinetics

• Structure determination of RPLP1-containing human ribosomes: High-resolution structures would provide mechanistic insights into how RPLP1 influences ribosome function

• Tissue-specific, inducible RPLP1 modulation in vivo: More sophisticated genetic tools for manipulating RPLP1 expression with spatial and temporal precision would clarify its tissue-specific functions

These technological developments would address current limitations in understanding RPLP1's precise mechanisms of action.

How might RPLP1 research inform broader understanding of specialized ribosomes?

RPLP1 research has significant implications for the emerging field of specialized ribosomes:

• RPLP1's "fine-tuning" of translation without affecting global protein synthesis provides evidence for specialized ribosome functions

• Understanding how RPLP1 selectively influences specific protein subsets could reveal mechanisms by which ribosomal composition affects translational preferences

• RPLP1's essential role in development suggests specialized ribosomes may be particularly important during developmental processes

• The tissue-specific consequences of RPLP1 dysfunction indicate that specialized ribosomes may have particularly critical roles in certain tissues

• RPLP1's involvement in the ribosomal stalk suggests this structure may be an important site for conferring specialized ribosomal functions

By elucidating RPLP1's precise molecular mechanisms, researchers can gain insights into how variations in ribosomal composition contribute to specialized functions across tissues, developmental stages, and disease states.