Recombinant 60S ribosomal protein L36 (rpl-36)

How does RPL36 contribute to ribosome structure and function?

As a component of the 60S ribosomal subunit, RPL36 contributes to the structural integrity of the ribosome. Ribosomes consist of a small 40S subunit and a large 60S subunit, which together comprise four RNA species and approximately 80 structurally distinct proteins . RPL36 participates in the assembly of the 60S subunit and helps maintain its three-dimensional structure. This structural role is essential for proper ribosome function, including mRNA binding, translation initiation, and peptide bond formation during protein synthesis. Experimental approaches to study these contributions include cryo-electron microscopy, X-ray crystallography, and functional assays that assess translation efficiency in the presence of RPL36 mutations or deletions.

What is alt-RPL36 and how does it differ from canonical RPL36?

Alt-RPL36 is a 148-amino acid protein co-encoded with human RPL36, discovered through proteomic strategies for identifying unannotated short open reading frames . Unlike canonical RPL36, which is part of the ribosome structure, alt-RPL36 is expressed from an upstream non-ATG start codon (GTG) in the RPL36 transcript variant 2 . Alt-RPL36 functions independently of canonical RPL36 in cellular signaling rather than directly in ribosome structure. The protein partially localizes to the endoplasmic reticulum where it interacts with TMEM24, a protein involved in phosphatidylinositol transport . This represents an important example of a human transcript expressing two sequence-independent polypeptides from overlapping open reading frames (ORFs) that regulate the same processes via distinct mechanisms .

What experimental approaches are used to distinguish between RPL36 and alt-RPL36 in research?

Distinguishing between RPL36 and alt-RPL36 requires specific experimental approaches:

• Epitope tagging: Researchers append myc, FLAG, or HA tags to the proteins for differential detection. For example, dual FLAG and HA tags have been used at the 3' end of alt-RPL36 for stable expression studies .

• Start codon manipulation: The GTG start codon of alt-RPL36 can be mutated to ATG to enhance expression for experimental purposes, which helps differentiate it from canonical RPL36 .

• Fluorescent labeling techniques: Methods such as inverse-electron-demand Diels-Alder cycloaddition with tetrazine-Cy5 have been employed to fluorescently label alt-RPL36, followed by confocal microscopy imaging .

• Specific antibodies: Development of antibodies that recognize unique epitopes in each protein enables direct detection through Western blotting and immunofluorescence.

• APEX fingerprinting: This proximity labeling technique has been used with APEX2-fusion proteins to identify the distinct interactomes of RPL36 and alt-RPL36 .

How does alt-RPL36 regulate the PI3K-AKT-mTOR signaling pathway?

Alt-RPL36 functions as a negative regulator of the PI3K-AKT-mTOR signaling pathway through its interaction with TMEM24 at the endoplasmic reticulum. TMEM24 is responsible for transporting the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane . Through knockout experiments, researchers have demonstrated that:

• Deletion of alt-RPL36 increases PI(4,5)P2 levels in the plasma membrane

• This elevation in PI(4,5)P2 upregulates the PI3K-AKT-mTOR signaling pathway

• The signaling changes ultimately lead to increased cell size

This regulatory role is distinct from the canonical function of RPL36 in ribosome composition, providing a fascinating example of how a single gene can encode two functionally distinct proteins that regulate cellular growth through different mechanisms .

What is the significance of alt-RPL36 phosphorylation in cellular signaling?

Alt-RPL36 contains four serine residues (S19, S22, S140, and S142) that undergo phosphorylation, making it the first example of a phosphorylated alternative ORF product . The phosphorylation status of these serines is crucial for alt-RPL36 function:

• Mutation of all four serines to alanine abolishes the interaction with TMEM24

• This loss of interaction prevents alt-RPL36 from regulating PI3K signaling

• The phosphorylation appears hierarchical, with pS22 required for phosphorylation of S19

• S140 and S142 phosphorylation occurs independently, with both sites contributing to the third band observed in gel electrophoresis

Experimental evidence indicates that approximately 65% of alt-RPL36 exists in phosphorylated forms under standard growth conditions, suggesting dynamic regulation of this modification. The phosphorylation status directly impacts alt-RPL36's ability to regulate cell size and growth through the PI3K-AKT-mTOR pathway .

What are the recommended methods for recombinant RPL36 expression and purification?

Recombinant RPL36 can be expressed and purified using several established protocols:

• Expression System: E. coli is commonly used for RPL36 expression, as seen in the production of recombinant protein antigens .

• Fusion Tags: N-terminal His6 tags facilitate purification, while additional tags like Albumin Binding Protein (ABP) derived from Streptococcal Protein G can enhance stability .

• Purification Method: Immobilized metal affinity chromatography (IMAC) is typically employed for purification, yielding concentrations greater than 0.5 mg/ml .

• Buffer Conditions: PBS with 1M Urea at pH 7.4 has been used for maintaining protein stability without preservatives .

• Storage Recommendations: Storage at -20°C with avoidance of freeze-thaw cycles maximizes stability .

For alt-RPL36, expression typically involves cloning into vectors like pcDNA3 for transient expression or pLJM1 for stable expression, with epitope tags (myc, FLAG, HA) appended to the 3' end to facilitate detection and purification .

What gene editing approaches are most effective for studying RPL36 and alt-RPL36 function?

Several gene editing approaches have proven effective for studying RPL36 and alt-RPL36:

• CRISPR-Cas9 Knockout: Complete knockout of the gene allows assessment of loss-of-function phenotypes, as demonstrated in HEK 293T cells where alt-RPL36 knockout increased PI(4,5)P2 levels and upregulated PI3K-AKT-mTOR signaling .

• Point Mutation Generation: Site-directed mutagenesis to create specific mutations, such as the serine-to-alanine mutations in alt-RPL36, enables structure-function studies of particular amino acids .

• Start Codon Modification: Altering the GTG start codon of alt-RPL36 to ATG enhances expression for experimental purposes while maintaining function .

• Frame-Specific Editing: Selective modification of one reading frame while leaving the other intact allows examination of RPL36 and alt-RPL36 functions independently.

• Inducible Expression Systems: Doxycycline-inducible systems permit temporal control over gene expression, useful for studying dynamic processes.

When designing gene editing experiments, researchers should consider the potential impact on both reading frames to avoid unintended consequences on the expression of either RPL36 or alt-RPL36.

How can researchers investigate the distinct yet complementary roles of RPL36 and alt-RPL36 in regulating translation?

To investigate the distinct yet complementary roles of RPL36 and alt-RPL36 in regulating translation, researchers can employ several sophisticated approaches:

• Polysome Profiling: This technique separates actively translating ribosomes on sucrose gradients to assess global translation rates in cells with manipulated RPL36 or alt-RPL36 expression.

• Ribosome Footprinting: This method identifies ribosome-protected mRNA fragments through next-generation sequencing to determine translation efficiency of specific mRNAs under different conditions.

• Bicistronic Reporter Assays: Custom-designed reporters containing both reading frames allow simultaneous monitoring of RPL36 and alt-RPL36 expression and their effects on downstream targets.

• Quantitative Phosphoproteomics: Mass spectrometry-based approaches to quantify changes in phosphorylation status of signaling proteins in the PI3K-AKT-mTOR pathway following manipulation of alt-RPL36.

• Live-Cell Imaging: Fluorescently labeled PI(4,5)P2 sensors combined with fluorescently tagged alt-RPL36 enable real-time visualization of phospholipid dynamics and protein localization.

What are the implications of RPL36 and alt-RPL36 for understanding multicistronic gene expression in humans?

The co-expression of RPL36 and alt-RPL36 from a single transcript has significant implications for understanding multicistronic gene expression in humans:

• Prevalence of Polycistronic Genes: The RPL36 example suggests that multicistronic gene arrangements may be more common in humans than previously recognized, with up to 30% of alternative ORFs overlapping annotated protein-coding sequences in different reading frames .

• Evolutionary Conservation: Investigating the conservation of this dual-coding arrangement across species may reveal evolutionary pressures that maintain functionally distinct proteins from a single transcript.

• Regulatory Networks: This arrangement allows for coordinated expression of proteins that regulate the same cellular processes through different mechanisms, as exemplified by RPL36 and alt-RPL36 both influencing protein synthesis but via distinct pathways .

• Non-ATG Initiation: The use of a GTG start codon for alt-RPL36 highlights the importance of near-cognate non-AUG start codons, which may initiate up to 50% of unannotated alternative ORFs .

• Post-Transcriptional Regulation: Exploring how translation efficiency is balanced between the two reading frames may reveal novel mechanisms of post-transcriptional regulation.

The RPL36/alt-RPL36 example expands our understanding of genomic complexity and suggests that thousands of previously unannotated functional proteins may exist in the human proteome, requiring advances in computational prediction and experimental validation methods .

What are common challenges in detecting and studying RPL36 and alt-RPL36, and how can they be overcome?

Researchers face several challenges when studying RPL36 and alt-RPL36:

Researchers should also be aware that stabilizing recombinant RPL36 often requires specific buffer conditions (PBS with 1M Urea at pH 7.4) and storage at -20°C to avoid freeze-thaw cycles that can compromise protein integrity .

How can researchers effectively measure the impact of RPL36 and alt-RPL36 on the PI3K-AKT-mTOR signaling pathway?

To effectively measure the impact of RPL36 and alt-RPL36 on the PI3K-AKT-mTOR signaling pathway, researchers can employ the following methodological approaches:

• Phosphorylation Status Analysis:

  • Western blotting with phospho-specific antibodies against key pathway components (p-AKT, p-mTOR, p-S6K, p-4EBP1)

  • Flow cytometry for single-cell quantification of phosphorylated proteins

  • Reverse-phase protein arrays for high-throughput phosphoprotein profiling

• PI(4,5)P2 Level Measurement:

  • Immunofluorescence microscopy with PI(4,5)P2-specific antibodies

  • Biochemical extraction and quantification of phospholipids by mass spectrometry

  • Live-cell imaging with fluorescent PI(4,5)P2 sensors

• Cell Size and Growth Assays:

  • Forward scatter analysis by flow cytometry to measure cell size

  • Automated cell counting and size determination using platforms like Coulter counter

  • Time-lapse microscopy for tracking growth rates in real-time

• Functional Pathway Analysis:

  • Drug inhibition studies using selective inhibitors of PI3K, AKT, or mTOR

  • Genetic epistasis experiments combining alt-RPL36 manipulation with pathway component knockdowns

  • Rescue experiments with constitutively active pathway components

• TMEM24 Interaction Studies:

  • Co-immunoprecipitation with wild-type and mutant alt-RPL36 variants

  • FRET or BiFC analysis for real-time interaction monitoring

  • In vitro phospholipid transfer assays measuring TMEM24 activity

These methodologies can be combined to comprehensively assess how alt-RPL36, particularly through its phosphorylation status and interaction with TMEM24, regulates the PI3K-AKT-mTOR signaling pathway and ultimately affects cell growth and size .