Recombinant Pongo pygmaeus 40S ribosomal protein S4, Y isoform 1 (RPS4Y1)

What is the evolutionary origin of RPS4Y1?

RPS4Y1 emerged through a duplication event that occurred after the divergence of New World monkeys, approximately 35 million years ago. The evolutionary timeline has been established through DNA sequence analysis of intronic and cDNA regions of RPS4Y genes across the primate phylogeny . Prior to this duplication, a single ancestral RPS4 gene existed, which later diverged into X-linked and Y-linked versions. In the primate lineage, a second duplication event led to RPS4Y2, creating a gene family with three members: RPS4X, RPS4Y1, and RPS4Y2 . This evolutionary pattern is significant as it reflects adaptation processes unique to primate species.

The evolutionary analysis reveals distinct clustering of RPS4X, RPS4Y1, and RPS4Y2 sequences across species, with pseudogene sequences generally grouping with RPS4Y1 clusters. This suggests that while RPS4Y1 maintains essential functions, it has evolved under different selective pressures compared to its paralogues .

How does RPS4Y1 expression differ between males and females?

RPS4Y1 exhibits strong male-specific expression patterns, making it a reliable sex-specific marker in gene expression datasets. As a Y-chromosome linked gene, RPS4Y1 is only expressed in males, while RPS4X is expressed in both sexes . The probe set 201909_at for RPS4Y1 shows consistently high expression values in male samples and minimal expression in female samples across diverse tissue types and disease states .

This sex-specific expression pattern has been utilized in bioinformatic applications for sample annotation error detection in genomic studies. After normalization procedures, RPS4Y1 expression can reliably distinguish between male and female samples with high accuracy . The expression difference is so distinctive that it can be used alongside other sex-specific genes (like DDX3Y, XIST) to verify sample sex annotations in genomic datasets.

What is the structural composition of RPS4Y1 protein?

RPS4Y1 is a component of the 40S ribosomal subunit, sharing substantial structural homology with RPS4X. The protein contains several functional domains, including a KOW motif that is important for RNA binding and ribosomal function . Sequence analysis has identified several critical amino acid positions that may influence protein function, including position 68, 70, 87, 108, and 185 .

Comparative analyses between human and other primate RPS4Y1 have identified specific amino acid replacements that may influence protein function. Particularly significant is a change in the KOW protein domain that affects an invariable position in this motif, suggesting potential functional consequences . These structural features are important considerations when working with recombinant RPS4Y1 proteins.

What evidence exists for positive selection in RPS4Y1 evolution?

Maximum likelihood analyses of synonymous and non-synonymous substitutions have revealed that RPS4Y2, but not RPS4Y1, underwent positive selection specifically in the human lineage . This represents the first evidence of positive selection acting on a ribosomal protein gene. When applying the one-ratio (M0) model and free-ratio (FR) model to RPS4Y2 sequences, the FR model fit the data significantly better, indicating variable selective pressure across branches .

The evidence for positive selection in RPS4Y2 was particularly strong in the human lineage. The branch-site models identified several amino acid positions under positive selection, as shown in the following table:

The table indicates specific amino acid residues under positive selection in human RPS4Y2 (Hsa Y2) . Although this data pertains primarily to RPS4Y2, it provides important evolutionary context for understanding RPS4Y1 function and divergence.

What is the role of RPS4Y1 in placental development and preeclampsia?

Research has demonstrated that RPS4Y1 is aberrantly highly expressed in preeclamptic placentae . Mechanistically, RPS4Y1 restricts the migration and invasion of trophoblast cells by downregulating STAT3 (signal transducer and activator of transcription 3) phosphorylation . This RPS4Y1-STAT3 axis subsequently influences the downstream epithelial-mesenchymal transition pathway in trophoblast cells, which is crucial for proper placental development .

The dysregulation of RPS4Y1 in preeclampsia represents the first report linking this ribosomal protein to a pregnancy complication. The aberrant expression of RPS4Y1 and reduced STAT3 phosphorylation correlate with the clinical manifestation of preeclampsia, suggesting a potential pathophysiological mechanism . This discovery opens new avenues for understanding sex-linked genetic factors in pregnancy complications and may offer insights into the development of diagnostic or therapeutic strategies.

How do RPS4Y1 and RPS4X functionally complement each other?

Despite their location on different sex chromosomes, RPS4Y1 and RPS4X appear to be functionally interchangeable but both necessary for proper development . RPS4X escapes X-inactivation in females, suggesting its essential function. In males, both RPS4X (from the X chromosome) and RPS4Y1 (from the Y chromosome) contribute to ribosome assembly and function, although RPS4Y1 is expressed at lower levels .

Sequence analyses have shown that RPS4Y1 has evolved under fewer functional constraints compared to RPS4X, as evidenced by an increased substitution rate in great ape RPS4Y1 compared to the X-linked copies . This differential evolution suggests that while both proteins maintain essential ribosomal functions, they may have developed subtle functional differences that contribute to sex-specific cellular processes.

What techniques are effective for studying RPS4Y1 expression levels?

Several methodological approaches have been validated for studying RPS4Y1 expression. Microarray analysis using specific probe sets (such as 201909_at) has demonstrated high reliability in detecting RPS4Y1 expression . For more precise quantification, quantitative PCR with gene-specific primers is recommended, particularly when examining expression across different tissues or experimental conditions.

When analyzing RPS4Y1 expression across multiple datasets, normalization is critical to account for platform-specific variations. A proven approach involves a linear transformation to median values of 0 and 1 for low and high expression groups, respectively . This normalization allows for reliable comparison of RPS4Y1 expression patterns across different cohorts and experimental platforms.

For classification of samples based on RPS4Y1 expression, a robust statistical approach involves:

• Clustering expression values into low and high groups

• Fitting a normal distribution to the low expression group

• Setting a cut-point at the 99.9% quantile of the fitted distribution

• Classifying samples above this threshold as belonging to the high expression group

This methodology has proven effective for sample annotation verification in genomic studies.

What experimental designs are optimal for studying RPS4Y1 function in trophoblast cells?

Based on published research on RPS4Y1's role in trophoblast cell migration and invasion, several experimental approaches are recommended:

• Gene expression modulation studies using overexpression constructs or RNA interference to alter RPS4Y1 levels in trophoblast cell lines

• Migration and invasion assays (e.g., wound healing, transwell, and Matrigel invasion assays) to assess the impact of RPS4Y1 modulation on trophoblast cell behavior

• Western blot analysis to monitor STAT3 phosphorylation status in response to RPS4Y1 manipulation

• Immunohistochemistry of placental tissues to compare RPS4Y1 expression between normal and preeclamptic placentae

• Co-immunoprecipitation experiments to identify protein interaction partners of RPS4Y1 in trophoblast cells

These experimental approaches should be accompanied by appropriate controls, including comparison with RPS4X expression and function to distinguish Y-linked specific effects from general ribosomal protein functions .

How can researchers effectively produce and purify recombinant Pongo pygmaeus RPS4Y1?

For producing recombinant Pongo pygmaeus (orangutan) RPS4Y1, researchers should consider the following methodological approach:

• Gene synthesis based on the published orangutan RPS4Y1 sequence or PCR amplification from genomic DNA

• Cloning into an appropriate expression vector with a purification tag (e.g., His-tag, GST-tag)

• Expression in a eukaryotic system (preferred) or bacterial system with careful optimization of induction conditions

• Purification using affinity chromatography followed by size exclusion chromatography to ensure protein homogeneity

• Validation of protein folding and activity through functional assays

When working with recombinant RPS4Y1, it's important to consider its RNA-binding properties. The literature suggests that both CBX and HPH proteins, which interact with components in the polycomb repressive complex, demonstrate RNA binding capacity . This might be relevant for RPS4Y1 as well, given its role in ribosomal function.

What are the implications of RPS4Y1 in other disease models beyond preeclampsia?

Given the ubiquitous expression of RPS4Y1 in male tissues and its role in fundamental cellular processes, investigating its involvement in other disease models represents an important research direction. Researchers should consider examining RPS4Y1 expression and function in male-specific disorders, sex-biased conditions, and diseases involving ribosomal dysfunction.

The observation that RPS4Y1 influences STAT3 signaling suggests potential implications in inflammatory and immune-related conditions, as STAT3 is a critical mediator of various cytokine signaling pathways. Further studies could explore how RPS4Y1 expression correlates with disease progression or treatment response in conditions with known STAT3 involvement.

How do species differences in RPS4Y1 affect experimental model selection?

The evolutionary analysis of RPS4Y1 across primate species reveals important considerations for experimental model selection. The duplication event that generated RPS4Y2 occurred after the divergence of New World monkeys , meaning that species like marmosets and squirrel monkeys only possess a single RPS4Y gene.

When selecting animal models for RPS4Y1 research, researchers should consider these evolutionary differences. Great apes (chimpanzees, gorillas, orangutans) share the most similar RPS4Y1/Y2 gene structure with humans, while Old World monkeys (macaques, baboons) possess both genes but show different evolutionary patterns . The following comparative approach is recommended:

• Clearly identify the evolutionary status of RPS4Y1 in the selected model organism

• Account for species-specific amino acid differences, particularly at positions identified under positive selection

• Consider the potential functional divergence between human and model organism RPS4Y1

• When possible, validate findings across multiple species to distinguish conserved from species-specific functions