Recombinant Macaca fuscata fuscata 40S ribosomal protein S4, Y isoform 1 (RPS4Y1)

What is the basic structure and function of RPS4Y1 in Macaca fuscata fuscata?

RPS4Y1 is a Y-chromosome-encoded ribosomal protein involved in mRNA binding and ribosome assembly. As a core component of both 40S and 60S ribosomal subunits, it plays a critical role in translational fidelity and ribosome biogenesis. The protein contains 263 amino acids with a molecular weight of approximately 29 kDa and features several functional domains including S4, Ribosomal-S4e, and KOW motifs that facilitate RNA interactions and structural stability. The KOW motif specifically contributes to RNA binding capabilities while the S4e domains ensure proper integration into ribosomal subunits, enabling accurate translation of genetic information into proteins.

How does RPS4Y1 differ from its X-linked homolog RPS4X in primates?

Despite being encoded by different chromosomes, RPS4Y1 and RPS4X are functionally equivalent and interchangeable in ribosome assembly . Both contain 263 amino acids with conserved RNA-binding domains, though they exhibit sequence divergence. RPS4X is highly conserved across primates with approximately 98% amino acid identity between humans and Macaca fuscata, while RPS4Y1 shows greater evolutionary flexibility with increased substitution rates in great apes compared to its X-linked counterpart . RPS4X escapes X-inactivation in placental mammals, allowing both proteins to be present in male ribosomes, whereas only RPS4X is present in female ribosomes . This arrangement suggests complementary roles in ribosome function despite their distinct evolutionary trajectories.

What is the evolutionary history of RPS4Y1 in primates?

RPS4Y1 evolved through a gene duplication event that occurred after the divergence of New World monkeys, approximately 35 million years ago . Following this event, a second Y-linked paralog (RPS4Y2) emerged. The Macaca fascicularis group, which includes M. fuscata, originated from hybridization between sinica and silenus macaque groups approximately 3.45-3.56 million years ago. During this hybridization event, these macaques inherited Y chromosomes (including RPS4Y1) from the silenus lineage, while mitochondrial DNA derived from sinica ancestors. The conservation of RPS4Y1 in hybrid lineages suggests its essential role in maintaining ribosomal function despite genomic mosaicism resulting from hybridization.

What are the recommended methods for expression and purification of recombinant RPS4Y1?

For efficient expression and purification of recombinant Macaca fuscata RPS4Y1, researchers should consider a multi-step approach. First, clone the RPS4Y1 cDNA into a bacterial expression vector containing an N-terminal His-tag for purification purposes. Expression in E. coli BL21(DE3) cells at lower temperatures (16-18°C) after IPTG induction helps minimize inclusion body formation. Purification typically involves immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by ion-exchange chromatography to remove contaminants, and size-exclusion chromatography to obtain homogeneous protein. For functional studies, it's crucial to verify proper folding through circular dichroism spectroscopy. When designing expression constructs, consideration of the S4e and KOW domains (positions 1-80 and 81-150, respectively) is essential for maintaining RNA-binding capability in the recombinant protein.

How can researchers effectively analyze interactions between RPS4Y1 and other ribosomal components?

To analyze interactions between RPS4Y1 and other ribosomal components, researchers should employ a combination of biochemical and structural techniques. Pull-down assays using tagged recombinant RPS4Y1 can identify direct binding partners within ribosomal extracts. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) provide quantitative binding parameters for interactions with specific rRNAs or proteins. Crosslinking followed by mass spectrometry (XL-MS) can map interaction interfaces within assembled ribosomes. For structural characterization, cryo-electron microscopy of reconstituted ribosomal subunits containing recombinant RPS4Y1 offers insights into integration within the 40S subunit. Additionally, comparative analyses between RPS4Y1 and RPS4X using these techniques can reveal functional equivalence or subtle differences in binding preferences despite their sequence divergence, which is particularly relevant given their interchangeability in ribosome assembly.

What functional assays are available to assess the biological activity of recombinant RPS4Y1?

To assess the biological activity of recombinant RPS4Y1, researchers can employ several complementary functional assays. In vitro translation assays using rabbit reticulocyte lysate or wheat germ extract systems supplemented with recombinant RPS4Y1 can evaluate its capacity to support protein synthesis. Ribosome assembly assays tracking the incorporation of labeled RPS4Y1 into 40S subunits provide insights into structural integration capabilities . For cellular studies, complementation assays in RPS4Y1-depleted cells can assess whether the recombinant protein restores normal translation rates and fidelity. Given RPS4Y1's role in preeclampsia, trophoblast cell migration and invasion assays represent disease-relevant functional readouts, with STAT3 phosphorylation serving as a molecular marker of activity . Researchers should include parallel assays with recombinant RPS4X to determine functional equivalence, and consider species-specific controls when working with macaque proteins in human cell systems.

How does RPS4Y1 contribute to sex-specific differences in translation and development?

RPS4Y1 contributes to sex-specific differences in translation through its exclusive expression in males, where it accounts for approximately 15% of male ribosomes, working alongside the ubiquitously expressed RPS4X. This creates sexually dimorphic ribosome populations that may influence translational dynamics in a sex-specific manner. To investigate these differences experimentally, researchers should implement ribosome profiling (Ribo-seq) comparing male cells with both RPS4Y1 and RPS4X to female cells with only RPS4X, analyzing translational efficiency across different mRNA subsets. Additionally, CRISPR-mediated knockout of RPS4Y1 in male cells followed by transcriptome and proteome analysis would reveal genes particularly sensitive to RPS4Y1 deficiency. Studies examining RPS4Y1's potential role in Turner syndrome suggest that haploinsufficiency of ribosomal protein S4 genes may contribute to developmental abnormalities, though this hypothesis remains controversial . Developmental timing assays in embryonic stem cell differentiation models could further elucidate how RPS4Y1 influences cell fate decisions during early development.

What is the role of RPS4Y1 in preeclampsia pathogenesis and how can recombinant protein studies advance this research?

RPS4Y1 has been implicated in preeclampsia pathogenesis through its effects on trophoblast cell function. Studies show that RPS4Y1 levels are upregulated in placental samples from preeclamptic patients compared to normotensive pregnant women . Mechanistically, RPS4Y1 appears to regulate trophoblast cell migration and invasion via the STAT3/epithelial-mesenchymal transition pathway, with inhibition of RPS4Y1 inducing trophoblast invasion and promoting placental explant outgrowth while increasing STAT3 phosphorylation . Researchers utilizing recombinant RPS4Y1 can advance this field by developing in vitro models where controlled introduction of the protein into trophoblast cultures allows dose-dependent assessment of invasion and migration. The following experimental approaches are recommended:

These approaches can provide deeper insights into how RPS4Y1 dysregulation contributes to the inadequate trophoblast invasion and impaired spiral artery remodeling characteristic of preeclampsia.

How can comparative studies of RPS4Y1 across primate species inform our understanding of ribosomal protein evolution?

Comparative studies of RPS4Y1 across primate species provide unique insights into ribosomal protein evolution under different selective pressures. The increased substitution rate observed in great ape RPS4Y1 compared to X-linked copies indicates fewer functional constraints on Y-linked genes . To leverage recombinant proteins for evolutionary studies, researchers should:

• Express and purify RPS4Y1 from multiple primate species representing key evolutionary branches (e.g., New World monkeys, Old World monkeys, great apes)

• Conduct ribosome reconstitution experiments to test functional interchangeability across species

• Perform in vitro translation assays comparing translation efficiency and fidelity

• Use structural biology approaches to identify amino acid changes that maintain function despite sequence divergence

Maximum likelihood analyses of synonymous and non-synonymous substitutions have revealed positive selection acting on the related RPS4Y2 gene in the human lineage - the first evidence of positive selection on a ribosomal protein gene . Similar analyses of RPS4Y1 across the primate phylogeny, complemented by functional studies with recombinant proteins, could identify adaptive changes responding to species-specific translational requirements or Y chromosome environments. The high conservation of RPS4X (98% amino acid identity between humans and Macaca fuscata) provides an excellent control for identifying Y-specific evolutionary patterns.

What are the major challenges in producing and studying recombinant RPS4Y1 and how can they be addressed?

Producing and studying recombinant RPS4Y1 presents several challenges that researchers should anticipate. First, ribosomal proteins often form insoluble aggregates when expressed in bacterial systems due to their highly charged surfaces and hydrophobic cores designed for integration into ribosomes. To address this, researchers should optimize expression conditions by using lower temperatures (16-18°C), specialized E. coli strains (e.g., Rosetta for rare codon usage), and fusion tags that enhance solubility (e.g., SUMO or MBP tags). Second, ribosomal proteins typically function as part of large complexes, making isolated functional studies challenging. This can be addressed by developing reconstitution systems where recombinant RPS4Y1 is incorporated into partial or complete ribosomal subunits. Third, proper folding verification is essential, as misfolded proteins may retain some binding capabilities while lacking full functionality. Circular dichroism spectroscopy and limited proteolysis can confirm structural integrity before functional assays. Finally, species-specific differences between macaque and human systems must be considered when designing experimental controls, particularly for cellular assays where human cell lines are commonly used.

How should researchers interpret contradictory data between RPS4Y1 and RPS4X functional studies?

When encountering contradictory data between RPS4Y1 and RPS4X functional studies, researchers should implement a systematic approach to investigation. First, validate protein quality by performing parallel experiments with independently produced protein batches to rule out preparation artifacts. Second, consider context-dependent functions by testing both proteins under identical experimental conditions while systematically varying buffer components, binding partners, or cellular environments to identify specific conditions where functional differences emerge. Third, conduct dose-response experiments, as apparent contradictions may reflect different activity thresholds rather than binary differences. Fourth, examine species specificity, as contradictions may arise when testing macaque proteins in human systems or vice versa.

For molecular mechanistic insights, researchers should compare the KOW motif and RNA-binding domains between RPS4Y1 and RPS4X through targeted mutagenesis, as these regions are critical for ribosomal function. The documented functional interchangeability between RPS4X and RPS4Y despite sequence divergence suggests robust structural conservation of key functional domains . When differences are consistently observed, researchers should consider tissue-specific or developmental stage-specific roles that may not be captured in standard assays. Publication of seemingly contradictory results is encouraged to advance understanding of the nuanced biological roles of these evolutionarily related proteins.

What considerations are important when designing experiments to study the role of RPS4Y1 in disease models?

When designing experiments to study RPS4Y1 in disease models, researchers must address several key considerations. First, establish appropriate dosage and expression levels that reflect physiological or pathological conditions, as both over- and under-expression can affect experimental outcomes. In preeclampsia studies, for example, RPS4Y1 upregulation in placental samples should be quantified and matched in experimental models . Second, select disease-relevant cellular contexts; for investigating preeclampsia, primary trophoblast cultures or choriocarcinoma cell lines are preferred over standard laboratory cell lines. Third, include functional readouts specific to the disease mechanism, such as trophoblast invasion assays and STAT3 phosphorylation status for preeclampsia research .

For genetic approaches, CRISPR-based strategies should target RPS4Y1 specifically without affecting RPS4X or RPS4Y2, which may require careful guide RNA design and validation. When introducing recombinant RPS4Y1 into cellular systems, researchers should consider creating stable cell lines with inducible expression to control protein levels precisely. For animal models, Y-chromosome editing presents unique challenges; therefore, xenograft models using manipulated human cells in immunocompromised animals may provide a more accessible alternative. Finally, researchers should design experiments that distinguish between RPS4Y1's canonical role in translation and potential specialized functions in disease contexts, potentially through complementation studies with RPS4X or mutant RPS4Y1 variants that selectively affect specific functions.

How might advanced structural biology techniques enhance our understanding of RPS4Y1 function?

Advanced structural biology techniques can significantly enhance our understanding of RPS4Y1 function by providing atomic-level insights into its interactions within the ribosome. Cryo-electron microscopy (cryo-EM) of intact ribosomes containing RPS4Y1 versus RPS4X would reveal subtle conformational differences that might influence translation dynamics. X-ray crystallography of isolated RPS4Y1 in complex with specific rRNA segments could identify critical contact points that determine functional specificity. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) would map conformational changes that occur upon RNA binding, while nuclear magnetic resonance (NMR) spectroscopy could characterize the dynamics of RPS4Y1's functional domains. These approaches would be particularly valuable for understanding how RPS4Y1's KOW motif (positions 81-150) interacts with RNA and how the S4e domains (positions 1-80 and 151-263) integrate into the ribosomal architecture. Researchers should focus on comparing macaque and human RPS4Y1 structures to identify species-specific features that might influence function in experimental systems, potentially explaining any observed differences in activity or binding properties.

What novel research questions could be addressed through comparative studies of RPS4Y1 and RPS4Y2?

Comparative studies of RPS4Y1 and RPS4Y2 present exciting opportunities to explore ribosomal protein specialization and evolution. Key research questions include: (1) How do the tissue-specific expression patterns of these paralogs (RPS4Y1 being ubiquitous versus RPS4Y2 being testis-specific) relate to functional specialization? (2) What structural adaptations in RPS4Y2 resulted from the positive selection identified in the human lineage, and how do these affect function? (3) Do ribosomes containing different RPS4 paralogs exhibit specialized translational properties for distinct mRNA subsets? To address these questions, researchers should develop paralog-specific antibodies and CRISPR knockout strategies to distinguish their individual contributions in cellular contexts.

Particularly promising approaches include ribosome profiling of cells expressing only RPS4Y1 versus RPS4Y2, structural studies comparing ribosome conformations with each paralog, and evolutionary analyses tracking selection pressures across primate lineages. The recent discovery of positive selection acting on RPS4Y2 represents the first evidence of positive selection on a ribosomal protein gene , suggesting that comparative functional studies may reveal unexpected specialized roles beyond the canonical function in translation. Understanding the evolutionary forces driving paralog diversification could provide broader insights into how ribosomal proteins adapt to sex-specific or tissue-specific translational environments.

How can integrative multi-omics approaches advance RPS4Y1 research in evolutionary and disease contexts?

Integrative multi-omics approaches can significantly advance RPS4Y1 research by connecting molecular function to broader biological impacts. In evolutionary contexts, researchers should combine genomic analyses of selection pressure with transcriptomics and proteomics to track how RPS4Y1 sequence changes correlate with species-specific gene expression patterns . This could reveal whether increased substitution rates in great ape RPS4Y1 influence translation of specific mRNA classes. For disease research, particularly preeclampsia where RPS4Y1 upregulation affects trophoblast function , multi-omics strategies should integrate:

• Transcriptomics: RNA-seq of normal versus preeclamptic placentas to identify differentially expressed genes

• Translatome analysis: Ribosome profiling to detect RPS4Y1-dependent changes in translation efficiency

• Proteomics: Quantitative protein analysis to confirm translational impacts

• Epigenomics: Chromatin accessibility and modification studies to understand RPS4Y1 regulation

• Metabolomics: Metabolite profiling to connect RPS4Y1 activity to cellular physiology

This integrated approach would provide a systems-level understanding of how RPS4Y1 dysregulation propagates through cellular networks in disease states. For example, the connection between RPS4Y1 and the STAT3/epithelial-mesenchymal transition pathway in preeclampsia could be expanded to identify additional signaling nodes and potential therapeutic targets. In evolutionary studies, comparative multi-omics across primate species would help explain why RPS4Y genes have been retained despite the presence of the functionally equivalent RPS4X, potentially revealing subtle specializations that conventional approaches have missed.