RPUSD4 Antibody

What is RPUSD4 and what cellular functions does it perform?

RPUSD4 is a mitochondrial pseudouridine synthase that catalyzes uridine to pseudouridine isomerization of various mitochondrial RNA substrates . It acts specifically on position 1397 in 16S mitochondrial ribosomal RNA (16S mt-rRNA), a modification essential for the assembly of 16S mt-rRNA into functional mitochondrial ribosomes . RPUSD4 also pseudouridylates position 39 in mitochondrial tRNA(Phe) . Recent studies have shown that RPUSD4 functions within a protein-RNA module that includes RCC1L, NGRN, RPUSD3, TRUB2, FASTKD2, and 16S mt-rRNA, which collectively controls 16S mt-rRNA abundance and is required for intra-mitochondrial translation . Notably, RPUSD4 also acts in the nucleus to regulate pre-mRNA splicing through pseudouridylation of pre-mRNAs at locations associated with alternatively spliced regions .

How can I validate the specificity of RPUSD4 antibodies in my experimental system?

Validating antibody specificity is crucial for reliable results. For RPUSD4 antibodies, implement the following validation strategy:

• Positive controls: Use cell lines known to express RPUSD4 (such as 143B cells described in the literature) .

• Knockdown verification: Perform siRNA or shRNA-mediated knockdown of RPUSD4 (approximately 80% reduction has been achieved in published studies) and confirm reduced signal in Western blots .

• Subcellular fractionation: Verify mitochondrial localization through subcellular fractionation and alkaline sodium carbonate extraction. RPUSD4 should appear in the mitochondrial fraction but not be associated with membranes .

• Proteinase K protection assay: Since RPUSD4 is a matrix protein, it should be protected from proteinase K digestion in intact mitochondria and mitoplasts but digested in permeabilized mitochondria .

• Band size verification: Confirm the expected molecular weight (approximately 43 kDa for human RPUSD4) .

A methodological note: When performing Western blotting, use 25 μg of protein per lane, block with 3% nonfat dry milk in TBST, and use antibody dilutions of 1:200-1:2000 (polyclonal) or according to manufacturer's recommendations for optimal results .

What are the optimal fixation and permeabilization conditions for RPUSD4 immunocytochemistry?

For successful immunocytochemical detection of RPUSD4:

• Fixation: Since RPUSD4 is primarily found in mitochondrial RNA granules (MRGs), use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve mitochondrial structure.

• Permeabilization: Use 0.2-0.5% Triton X-100 for 10 minutes to allow antibody access to mitochondrial proteins without disrupting mitochondrial integrity.

• Co-staining: For validation, co-stain with established mitochondrial markers (such as TOMM20 for outer membrane or HSP60 for matrix) and RNA granule markers.

• Controls: Include cells where RPUSD4 has been silenced through shRNA as negative controls .

• Antibody dilution: Start with 1:100-1:200 dilutions for monoclonal antibodies in ICC/IF applications .

The critical step is achieving sufficient permeabilization to detect this matrix protein while maintaining mitochondrial morphology for accurate localization to mitochondrial RNA granules.

How can I investigate RPUSD4's interaction with mitochondrial RNAs?

To study RPUSD4's interaction with its RNA substrates:

• RNA-immunoprecipitation (RIP): Use anti-RPUSD4 antibodies for immunoprecipitation followed by RT-PCR or RNA-seq to identify bound RNAs. Research has identified 16S mt-rRNA, mt-tRNA Met, and mt-tRNA Phe as binding partners .

• Cross-linking immunoprecipitation (CLIP): For higher resolution mapping of binding sites, perform UV cross-linking before immunoprecipitation with RPUSD4 antibodies.

• Pseudouridylation site identification: To verify pseudouridylation activity, use CMC (N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) treatment followed by primer extension to identify specific pseudouridylation sites, such as position 1397 in 16S mt-rRNA or position 39 in mt-tRNA Phe .

• Sucrose gradient co-sedimentation: To analyze association with the mitochondrial ribosome large subunit, use isokinetic sucrose gradients followed by Western blotting with RPUSD4 antibodies and mitoribosomal marker proteins .

If RNA binding is not detected, ensure mitochondrial lysate conditions preserve RNA-protein interactions by avoiding high salt concentrations and using RNase inhibitors throughout the procedure.

What are the experimental approaches to study RPUSD4's role in mitochondrial ribosome assembly?

RPUSD4 is critical for mitochondrial large ribosomal subunit (mt-LSU) assembly. To investigate this function:

• Sucrose gradient analysis: After RPUSD4 knockdown, perform isokinetic sucrose gradient centrifugation to analyze the sedimentation profiles of mitoribosomal subunits. Published data shows that RPUSD4 co-sediments specifically with mt-LSU, and its depletion leads to reduced mt-LSU assembly .

• Quantitative proteomics: Compare the abundance of mitoribosomal proteins in control versus RPUSD4-depleted cells. Focus particularly on 39S mt-LSU proteins like MRPL45, MRPL11, MRPL3, and MRPL24, which show marked reduction upon RPUSD4 silencing .

• RNA analysis: Quantify 16S mt-rRNA levels using qRT-PCR, as RPUSD4 depletion leads to a significant decrease in 16S mt-rRNA abundance .

• Mitochondrial translation assay: Assess global mitochondrial translation by metabolic labeling with [35S]methionine and [35S]cysteine. RPUSD4-depleted cells show a marked decrease in mitochondrial protein synthesis .

• Respiratory function assessment: Measure oxygen consumption rate in permeabilized control and RPUSD4-silenced cells to connect ribosome dysfunction to physiological outcomes .

A critical control is to verify that mt-SSU (small subunit) proteins like DAP3 and MRPS27 remain unaffected, confirming specificity of RPUSD4's role in mt-LSU assembly .

How can I troubleshoot weak or nonspecific signals when using RPUSD4 antibodies in Western blots?

When encountering problems with RPUSD4 antibody performance in Western blots:

• Antibody dilution optimization: Test a range of antibody concentrations. For polyclonal antibodies, the recommended range is 1:200-1:2000; start at 1:1000 and adjust based on signal-to-noise ratio .

• Blocking optimization: Use 3% nonfat dry milk in TBST as the starting blocking buffer, but consider BSA-based blockers if background persists .

• Sample preparation: Ensure complete lysis of mitochondria, as RPUSD4 is a mitochondrial matrix protein that may require stronger lysis conditions than cytosolic proteins.

• Loading control selection: Use mitochondrial markers as loading controls rather than cytosolic housekeeping proteins to accurately normalize RPUSD4 levels.

• Exposure time adjustment: RPUSD4 detection may require longer exposure times (approximately 90 seconds has been reported) .

• Secondary antibody selection: Use HRP-conjugated anti-rabbit IgG at 1:10,000 dilution for optimal detection .

If nonspecific bands persist, consider pre-absorbing the antibody with cell lysate from RPUSD4-knockdown cells to reduce cross-reactivity.

How can RPUSD4 antibodies be used to investigate mitochondrial dysfunction in disease models?

RPUSD4's essential role in mitochondrial translation makes it relevant for studying mitochondrial diseases:

• Tissue analysis: Compare RPUSD4 expression and localization in normal versus diseased tissues using immunohistochemistry or Western blotting with validated antibodies .

• Cell models: Analyze RPUSD4 levels in cellular models of mitochondrial disease or after treatment with mitochondrial stressors.

• Functional correlation: Correlate RPUSD4 levels with mitochondrial function parameters:

  • OXPHOS subunit levels (by Western blotting)

  • Oxygen consumption rates

  • ATP production

  • Mitochondrial translation efficiency

Research has demonstrated that even partial depletion (approximately 50%) of RPUSD4 leads to reduced levels of respiratory complex subunits and decreased respiratory activity, suggesting RPUSD4 is a sensitive marker for mitochondrial dysfunction .

• Rescue experiments: In RPUSD4-depleted cells showing respiratory defects, reintroduce wild-type RPUSD4 to verify functional recovery, confirming the specificity of observed phenotypes .

When using patient samples, evaluate both protein levels and potential mutations in RPUSD4 that might affect its pseudouridylation activity or mitochondrial localization.

What considerations are important when studying RPUSD4's role in novel cell types or model organisms?

When expanding RPUSD4 research to new experimental systems:

• Sequence homology assessment: Before selecting antibodies, verify the sequence conservation of RPUSD4 epitopes in your model organism. The available antibodies have been validated for human, rat, and mouse samples .

• Antibody validation strategy:

  • For new cell types within validated species: Verify mitochondrial localization

  • For new species: Start with Western blot validation before attempting more complex applications

• Expression level considerations: RPUSD4 expression may vary across tissues and cell types. In tissues with high mitochondrial content (e.g., cardiac tissue, skeletal muscle), antibody dilutions may need adjustment.

• Knockout feasibility: Note that complete RPUSD4 knockout appears to be lethal in human cells, indicating its essential nature . Consider inducible or tissue-specific knockout strategies in animal models.

• Alternative approaches: For organisms where antibodies perform poorly, consider epitope tagging of endogenous RPUSD4 using CRISPR/Cas9, followed by detection with tag-specific antibodies.

When interpreting results, remember that RPUSD4's importance may vary across species depending on the relative significance of pseudouridylation in mitochondrial RNA processing in different evolutionary contexts.

What emerging research areas might benefit from RPUSD4 antibody applications?

Several frontier research areas could benefit from RPUSD4 antibody applications:

• Nuclear functions exploration: Recent findings suggest RPUSD4 acts in the nucleus on pre-mRNA splicing . Using antibodies for chromatin immunoprecipitation (ChIP) or nuclear/mitochondrial fractionation could reveal dual localization patterns and context-dependent functions.

• Stress response studies: Investigating how cellular stresses affect RPUSD4 localization, expression, and activity could reveal new regulatory mechanisms of mitochondrial translation adaptation.

• Cancer metabolism research: As mitochondrial translation is often dysregulated in cancer, RPUSD4 antibodies could help characterize altered mitochondrial ribosome assembly in tumors.

• Aging research: Since mitochondrial dysfunction is a hallmark of aging, studying RPUSD4 expression and function throughout the lifespan could provide insights into age-related translation defects.

• Drug development: RPUSD4 antibodies could be valuable tools for screening compounds that modulate mitochondrial ribosome assembly as potential therapeutics for mitochondrial diseases.

The development of phospho-specific antibodies against RPUSD4 would be particularly valuable to investigate potential regulatory post-translational modifications that may control its activity or localization in different cellular contexts.