RPL35A Human

What is RPL35A and what is its primary function in human cells?

RPL35A (also known as 60S ribosomal protein L35a, Cell growth-inhibiting gene 33 protein, or DBA5) is a component of the large 60S ribosomal subunit. It functions as an integral part of the ribosome, the large ribonucleoprotein complex responsible for protein synthesis in cells . The ribosome consists of a small 40S subunit and a large 60S subunit, with RPL35A being a critical component of the latter . As a ribosomal protein, RPL35A contributes to the structural integrity and functional capacity of the ribosome during translation. It has been demonstrated that RPL35A is required for the proliferation and viability of hematopoietic cells, highlighting its importance in blood cell development . The rat homolog of this protein is known to bind to both initiator and elongator tRNAs, suggesting that it is located at the P site, or both P and A sites, of the ribosome .

What is the genomic location and organization of the human RPL35A gene?

The human RPL35A gene is located at chromosome band 3q29-qter . Interestingly, the gene was initially incorrectly mapped to chromosome 18 using a PCR-based strategy with DNAs from human-rodent somatic cell hybrids . This mislocalization was later corrected through fluorescence in situ hybridization (FISH) analysis using a P1 clone containing the human intron-containing RPL35A gene . This accurate location was further confirmed by positive PCR analysis of a human-hamster somatic cell hybrid containing only human chromosome 3 .

Like many ribosomal protein genes, RPL35A has several processed pseudogenes spread throughout the genome . Additionally, transcript variants utilizing alternative transcription initiation sites and alternative polyadenylation signals exist for this gene . This genomic complexity creates challenges for researchers studying the functional gene versus its pseudogenes.

How are mutations in RPL35A associated with Diamond-Blackfan Anemia?

Diamond-Blackfan Anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by red blood cell aplasia. While mutations in several ribosomal protein genes (RPS19, RPS24, and RPS17) account for approximately one-third of DBA cases, RPL35A has been identified as another gene involved in DBA pathogenesis . The connection between RPL35A and DBA was established through a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray analysis of DBA patients with chromosome 3q deletions .

Research has demonstrated that RPL35A is essential for the maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal cell proliferation, and cell survival . Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines showed alterations in large ribosomal subunit rRNA processing in both RPL35A-mutated and some RPL35A wild-type cases . These findings established that haploinsufficiency (having only one functional copy) of large ribosomal subunit proteins, including RPL35A, contributes to bone marrow failure and potentially cancer predisposition in DBA patients .

Why does RPL35A deficiency specifically affect erythroid lineage development?

Despite being a ubiquitously expressed ribosomal protein, deficiencies in RPL35A predominantly affect the erythroid lineage in conditions like Diamond-Blackfan Anemia. Several mechanisms have been proposed to explain this lineage specificity:

• High Protein Synthesis Demands: Erythroid precursors have exceptionally high protein synthesis requirements, particularly for globin production during terminal differentiation. This may make them especially sensitive to defects in ribosome biogenesis and function caused by RPL35A mutations.

• Cell Proliferation Requirements: RPL35A has been specifically shown to be required for the proliferation and viability of hematopoietic cells . The rapid proliferation of erythroid progenitors during erythropoiesis may make them particularly vulnerable to deficiencies in this protein.

• Pre-rRNA Processing Defects: Research has demonstrated that RPL35A deficiency leads to alterations in large ribosomal subunit rRNA processing, specifically affecting the maturation of 28S and 5.8S rRNAs . These molecular defects may have a disproportionate impact on erythroid differentiation compared to other lineages.

Understanding these mechanisms is not only important for DBA pathophysiology but also provides insights into normal erythropoiesis and the role of ribosome biogenesis in cell lineage specification and differentiation.

What are the recommended detection methods for studying RPL35A expression?

Several methods can be employed to detect and study RPL35A expression in research settings:

• Western Blotting: Anti-RPL35A antibodies, such as rabbit polyclonal antibodies, have been validated for detecting RPL35A in human samples . This method allows visualization of the protein based on its molecular weight (~14.9 kDa). According to published data, antibodies have been successfully tested on various human cell lines including HeLa, HEK-293T, and Jurkat cells at concentrations of 0.04 μg/mL using 50 μg of whole cell lysate .

• Quantitative RT-PCR: This technique can be used to measure RPL35A transcript levels, as demonstrated in studies of DBA patient cell lines where decreased expression of RPL35A transcripts was confirmed by quantitative RT-PCR .

• Fluorescence In Situ Hybridization (FISH): This technique has been used to localize the RPL35A gene on chromosome 3q29-qter and could potentially be adapted to study RPL35A expression patterns in tissues .

• Recombinant Protein Analysis: Recombinant RPL35A protein can be used as a positive control in expression studies or for generating and validating antibodies .

When selecting a method, researchers should consider their specific research question, sample type, and available resources. For protein-level detection, Western blotting is often the method of choice, while for transcript-level analysis, qRT-PCR is preferred.

How should recombinant RPL35A protein be handled for experimental applications?

For researchers using recombinant RPL35A protein in their experimental workflows, proper handling and storage are essential to maintain protein integrity and functionality:

Storage Recommendations:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• For longer periods, store frozen at -20°C

• Avoid multiple freeze-thaw cycles

• For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA)

Formulation Details:

• Recombinant RPL35A is typically supplied as a sterile filtered clear solution

• Standard formulation includes 20 mM Tris-HCl buffer (pH 8.0), 0.4 M Urea, and 10% glycerol

• Protein concentration is typically around 0.5 mg/ml

Important Considerations:

• Recombinant RPL35A is often produced with a His-tag (commonly at the N-terminus), which may affect certain applications

• The denatured form of the protein (as typically supplied) is most suitable for Western Blot or imaging assays but may not be optimal for functional studies

• Researchers should be aware that recombinant proteins may not fully recapitulate all post-translational modifications present in the native protein

When using recombinant RPL35A for antibody validation, protein interaction studies, or as standards in quantitative assays, these handling guidelines will help ensure reliable and reproducible results.

What techniques are available for studying RPL35A in ribosome assembly and function?

Investigating the role of RPL35A in ribosome assembly and function requires specialized techniques:

• Ribosome Profiling: This technique provides genome-wide analysis of ribosome positioning on mRNAs, offering insights into translation dynamics. Applied to models with RPL35A deficiency, it could identify specific transcripts whose translation is most affected.

• Polysome Profiling: This method separates free ribosomal subunits, monosomes, and polysomes by sucrose gradient centrifugation. It can be used to assess the impact of RPL35A deficiency on global translation and ribosome assembly.

• RNA Analysis: Northern blotting, qRT-PCR, or RNA-seq targeting pre-rRNAs and mature rRNAs can reveal how RPL35A deficiency affects rRNA processing, as demonstrated in studies showing the requirement of RPL35A for maturation of 28S and 5.8S rRNAs .

• RNA Interference and CRISPR-Based Approaches: shRNA inhibition has been used to demonstrate that RPL35A is essential for 60S subunit biogenesis . CRISPR-Cas9 can be employed for more precise genetic manipulation.

• Patient-Derived Cell Lines: Lymphoblastoid cell lines from DBA patients with RPL35A mutations have been valuable for studying pre-rRNA processing defects associated with RPL35A haploinsufficiency .

• Protein-RNA Interaction Studies: Techniques such as RNA immunoprecipitation (RIP) or cross-linking immunoprecipitation (CLIP) could be used to investigate the interactions between RPL35A and specific RNAs during ribosome assembly.

These approaches provide complementary information about how RPL35A contributes to ribosome biogenesis and function, offering insights into both normal cellular processes and disease mechanisms.

How does RPL35A contribute to pre-rRNA processing and 60S subunit biogenesis?

RPL35A plays a critical role in ribosome biogenesis, particularly in the processing of precursor ribosomal RNAs (pre-rRNAs) and assembly of the 60S ribosomal subunit. Research using shRNA inhibition has demonstrated that RPL35A is essential for the maturation of 28S and 5.8S rRNAs, which are components of the large ribosomal subunit .

The process of ribosome biogenesis begins in the nucleolus with the transcription of ribosomal DNA into pre-rRNA, which undergoes a series of processing steps including cleavage, modification, and assembly with ribosomal proteins. RPL35A appears to be involved in specific aspects of this process, particularly in the later stages of 60S subunit maturation.

Analysis of pre-rRNA processing in Diamond-Blackfan Anemia patients with RPL35A mutations has shown alterations in large ribosomal subunit rRNA processing . This suggests that RPL35A deficiency disrupts normal pre-rRNA processing pathways, leading to defects in ribosome assembly that ultimately affect cellular functions dependent on protein synthesis.

The importance of RPL35A in these processes is underscored by the severe phenotypes observed when its expression or function is compromised, including impaired cell proliferation, reduced viability of hematopoietic cells, and disease manifestations such as the bone marrow failure characteristic of DBA .

What challenges exist in distinguishing the functional RPL35A gene from its pseudogenes?

Like many ribosomal protein genes, RPL35A has several processed pseudogenes spread throughout the genome, creating significant challenges for genomic analyses . These challenges include:

• Sequence Similarity: Pseudogenes often share high sequence homology with their functional counterparts, making it difficult to design unique primers or probes that specifically target the functional gene.

• Mapping Difficulties: As evidenced by the initial incorrect mapping of RPL35A to chromosome 18, the presence of pseudogenes can confound chromosome localization studies . The correct localization to chromosome 3q29-qter was only achieved through more specific techniques like FISH analysis .

• PCR-Based Analysis Complexities: Researchers have had to develop specialized strategies, such as PCR approaches that can distinguish intron-containing functional genes from intronless processed pseudogenes .

• Expression Analysis Complications: Some pseudogenes may be transcribed, further complicating expression studies. RNA-seq or qRT-PCR analyses need to be carefully designed to distinguish between transcripts from the functional gene versus pseudogenes.

• Mutation Screening Challenges: When screening for mutations in RPL35A associated with diseases like DBA, researchers must ensure they are analyzing the functional gene rather than pseudogenes to avoid false positives.

To overcome these challenges, researchers have employed various strategies, including using intron-spanning primers for PCR, focusing on intron sequences which are absent in processed pseudogenes, and employing FISH analysis with specific probes . These approaches have allowed for the correct localization of the functional RPL35A gene and enabled accurate mutation screening in patient populations.

What is known about potential extraribosomal functions of RPL35A?

While RPL35A is primarily characterized as a component of the 60S ribosomal subunit, growing evidence suggests that many ribosomal proteins have functions beyond their canonical roles in protein synthesis. Although the search results don't explicitly detail extraribosomal functions specific to RPL35A, several potential areas warrant investigation:

• Cell Proliferation Regulation: RPL35A has been shown to be required for the proliferation and viability of hematopoietic cells . The mechanism behind this requirement may involve both ribosomal and potential extraribosomal functions.

• Historical Nomenclature Clue: RPL35A was also known as "Cell growth-inhibiting gene 33 protein" (GIG33) , suggesting early observations of growth regulation functions that may extend beyond its role in the ribosome.

• Potential Stress Response Roles: Many ribosomal proteins are released from the nucleolus under stress conditions and participate in p53 regulation and other stress response pathways. RPL35A might have similar functions, particularly relevant to the pathophysiology of diseases like Diamond-Blackfan Anemia.

• RNA Binding Properties: The ability of the rat homolog to bind both initiator and elongator tRNAs suggests RNA-binding capabilities that could potentially be employed in contexts outside the ribosome.

• Disease-Specific Functions: The specific impact of RPL35A deficiency on erythroid development in DBA suggests possible lineage-specific functions that may involve interactions with erythroid-specific factors.

Further research using techniques such as proximity labeling, co-immunoprecipitation, or protein localization studies under various cellular conditions could help elucidate potential extraribosomal functions of RPL35A and their relevance to both normal physiology and disease states.

What emerging technologies might advance our understanding of RPL35A function?

Several cutting-edge technologies hold promise for deepening our understanding of RPL35A's roles in cellular processes:

• Cryo-Electron Microscopy (Cryo-EM): High-resolution structural studies could precisely locate RPL35A within the ribosome and reveal how it interacts with rRNAs, tRNAs, and other ribosomal proteins. This could help explain how mutations affect ribosome assembly and function.

• Ribosome Profiling and RNA-Seq: These approaches can provide genome-wide insights into how RPL35A deficiency affects translation of specific mRNAs, potentially explaining tissue-specific disease manifestations.

• CRISPR-Cas9 Genome Editing: Creating precise RPL35A mutations or conditional knockout models can help study its function in various contexts while avoiding the lethality of complete gene deletion.

• Single-Cell Analysis: Techniques like single-cell RNA-seq could reveal cell-type-specific responses to RPL35A deficiency and identify particularly sensitive cell populations, especially within the hematopoietic system.

• Proteomics and Interactomics: Advanced mass spectrometry and proximity labeling techniques could identify proteins that interact with RPL35A both within and outside the ribosome.

• Patient-Derived iPSCs: Induced pluripotent stem cells from DBA patients with RPL35A mutations could be differentiated into various lineages to study cell type-specific effects, particularly focusing on erythroid differentiation.

• Long-Read Sequencing: This technology could better distinguish between the functional RPL35A gene and its pseudogenes, as well as identify novel transcript variants.

These technological approaches, combined with computational methods for data integration and analysis, hold significant promise for advancing our understanding of RPL35A biology and its role in diseases like Diamond-Blackfan Anemia.

What are potential therapeutic implications of RPL35A research?

Research into RPL35A function and its role in diseases like Diamond-Blackfan Anemia could lead to several therapeutic approaches:

• Gene Therapy: For patients with RPL35A mutations, gene therapy approaches aimed at restoring normal RPL35A expression could potentially correct the underlying defect. This might involve delivery of the wild-type gene or correction of mutations using CRISPR-based approaches.

• Small Molecule Modulators: Compounds that enhance the function of the remaining wild-type RPL35A allele in haploinsufficient conditions or that stabilize partially functional mutant proteins could be therapeutic.

• Targeted Translation Enhancement: Understanding how RPL35A deficiency affects translation of specific mRNAs could lead to strategies that selectively enhance translation of key transcripts important for erythropoiesis.

• p53 Pathway Modulation: If RPL35A deficiency activates p53-dependent cell death in erythroid progenitors, temporary p53 inhibition during critical developmental windows might improve erythropoiesis.

• Cell Therapy Approaches: Patient-specific iPSCs with corrected RPL35A could potentially be differentiated into hematopoietic stem cells for autologous transplantation.

• Synthetic Biology Solutions: Engineered ribosomes or ribosome-inspired systems that can compensate for RPL35A deficiency represent a more speculative but potentially transformative approach.

While these therapeutic directions are largely hypothetical at present, they highlight how fundamental research on ribosomal proteins like RPL35A can ultimately lead to novel treatment strategies for currently incurable conditions such as Diamond-Blackfan Anemia.