FARSB Human

What is the molecular function of FARSB in human cells?

FARSB encodes one of the beta subunits of the phenylalanyl-tRNA synthetase (PheRS), a tetrameric enzyme composed of two alpha (FARSA) and two beta (FARSB) subunits. This enzyme catalyzes the attachment of phenylalanine to its cognate tRNA molecule, a critical step in protein synthesis . Methodologically, researchers can study this function through:

• In vitro aminoacylation assays measuring the catalytic activity of purified PheRS

• Structural analysis of the PheRS complex using X-ray crystallography or cryo-EM

• Cellular assays measuring protein synthesis rates following FARSB manipulation

The human phenylalanine-tRNA synthetase functions in the cytoplasm to ensure accurate translation of mRNA to protein, with FARSB being essential for proper enzymatic function .

How is FARSB expression regulated across different human tissues?

FARSB shows variable expression patterns across tissues, with particularly notable expression in liver cancer cells. According to analysis using multiple databases:

• TIMER database shows FARSB is significantly upregulated in hepatocellular carcinoma (HCC) and many other tumors compared to normal tissues

• HCCDB database confirms higher FARSB expression in HCC tissues compared to normal liver tissue

• TCGA data analysis revealed significantly higher FARSB expression in 377 HCC samples compared to 50 adjacent normal tissue samples

Methodologically, researchers typically use:

• RT-qPCR for quantitative mRNA expression analysis

• Western blotting and immunohistochemistry for protein expression

• RNA-seq for transcriptome-wide profiling

• Database mining through platforms like TIMER, GEPIA, and HCCDB

What experimental methods are most reliable for detecting FARSB protein?

For reliable FARSB protein detection, researchers employ multiple complementary techniques:

• Western blot analysis: Used to quantify FARSB protein levels in cell lines and tissue samples, as demonstrated in studies with Huh7 and MHCC97H cells

• Immunofluorescence analysis: Provides spatial information on FARSB localization within cells, used for co-localization studies with other proteins like Raptor

• Immunohistochemistry: Applied to tissue sections, including xenograft tumor tissues, to visualize FARSB expression patterns

• Co-immunoprecipitation: Utilized to confirm protein-protein interactions, such as between FARSB and Raptor

The Human Protein Atlas (HPA) database offers validated antibody methods for immunostaining of tissues and cell lines, providing comparative expression analysis of FARSB in normal and tumor tissues .

How do mutations in FARSB contribute to human neurodevelopmental disorders?

FARSB mutations have been linked to severe neurodevelopmental phenotypes with multisystem involvement. Research methodologies for investigating these relationships include:

• Whole Exome Sequencing (WES): Critical for identifying pathogenic variants, as demonstrated in cases of neurodegenerative disorder with diffuse brain calcifications

• Sanger sequencing: Used for confirmation and segregation analysis in families with suspected FARSB-related disorders

• Functional assays: To assess the impact of specific mutations on protein function

Key findings from clinical studies reveal:

• Homozygous pathogenic variants (e.g., FARSB: NM_005687.4:c.853G>A:p.E285K) have been identified in patients with neurodevelopmental disorders involving brain calcifications

• Compound heterozygosity for loss-of-function FARSB variants (p.Thr256Met and p.His496Lysfs*14) was identified in a patient with severe, lethal, multisystem developmental phenotype

• Expression studies in patient fibroblasts showed severe depletion of both FARSB and FARSA protein levels, indicating destabilization of total phenylalanyl-tRNA synthetase

• FARSB mutations result in multisystem clinical presentations including growth restriction, brain calcifications, and interstitial lung disease

What molecular mechanisms underlie FARSB's role in hepatocellular carcinoma progression?

FARSB has been implicated in hepatocellular carcinoma (HCC) through several mechanisms:

• mTORC1 pathway activation: FARSB activates the mTORC1 signaling pathway by suppressing Raptor phosphorylation

  • Gene Set Enrichment Analysis (GSEA) in TCGA-LIHC dataset showed significant enrichment of mTOR signaling pathway in FARSB overexpression groups

  • Western blot analysis demonstrated changes in mTORC1 activation levels following FARSB knockdown

• Regulation of cell proliferation and migration:

  • CCK-8 and EdU assays showed FARSB knockdown suppressed proliferation of HCC cells

  • Transwell and scratch tests demonstrated FARSB knockdown inhibited cell migration

  • Colony formation assays revealed decreased colony formation ability in FARSB knockdown cells

• Ferroptosis suppression:

  • FARSB suppresses ferroptosis in HCC cells by activating mTORC1 expression

  • Treatment with erastin (ferroptosis inducer) demonstrated differential effects based on FARSB expression levels

  • Western blot detection of GPX4 expression, ROS and MDA levels confirmed FARSB's role in ferroptosis regulation

• In vivo tumor promotion:

  • Xenograft tumor models showed smaller tumors with FARSB knockdown and larger tumors with FARSB overexpression

  • Rapamycin treatment could reverse the tumor-promoting effects of FARSB overexpression

How can FARSB serve as a prognostic biomarker in cancer research?

FARSB has emerged as a potential prognostic biomarker, particularly in hepatocellular carcinoma. Research approaches include:

• Survival analysis: Kaplan-Meier method to generate survival curves comparing high vs. low FARSB expression groups

• Cox regression models: For univariate and multivariate analysis of FARSB's relationship with clinical outcomes

• ROC curve analysis: To estimate the predictive ability of FARSB expression for one-year, three-year, or five-year survival

Key findings supporting FARSB's prognostic value:

Source: Univariate and multivariate Cox regression analysis from TCGA data

• High FARSB expression strongly correlates with poor prognosis in HCC patients (p<0.001)

• FARSB expression is significantly associated with tumor grade, stage, and TMN classification in the TCGA-LIHC dataset

• Multiple databases (TIMER, HCCDB, ICGC) consistently demonstrate FARSB upregulation in HCC

What experimental models are most effective for studying FARSB-related diseases?

Several experimental models have proven valuable for investigating FARSB functions and disease mechanisms:

• In vitro cellular models:

  • HCC cell lines (Huh7, MHCC97H) for studying FARSB's role in cancer progression

  • Patient-derived fibroblasts for investigating effects of FARSB mutations

  • Genetic manipulation approaches:

    • Lentiviral systems for FARSB knockdown or overexpression

    • CRISPR-Cas9 for gene editing and creating specific mutations

• In vivo models:

  • Xenograft tumor models in nude mice (BALB/c nu) to study FARSB's impact on tumor growth

  • Methodology includes subcutaneous inoculation of FARSB-modified HCC cells, tumor volume measurement, and subsequent histological analysis

  • Combined treatment approaches (e.g., rapamycin injection) to study pathway interactions

• Patient-derived samples:

  • Analysis of FARSB expression and mutations in clinical specimens through WES and Sanger sequencing

  • Tissue microarrays for high-throughput immunohistochemical analysis

How does FARSB interact with epigenetic mechanisms in human diseases?

FARSB function appears to be regulated by and potentially influences epigenetic mechanisms:

• DNA methylation patterns:

  • UALCAN analysis revealed differential methylation of FARSB promoter in HCC compared to normal tissues

  • FARSB has been identified as a hypomethylated marker in HCC, suggesting epigenetic regulation of its expression

• Relationship with m6A modification:

  • FARSB may provide clues about m6A modification in HCC

  • Research connections with m6A-related genes including LRPPRC, RBM15B, and HNRNPA2B1 have been explored using GEPIA

• Methodological approaches:

  • Bisulfite sequencing for DNA methylation analysis

  • MeRIP-seq (Methylated RNA immunoprecipitation sequencing) for m6A modification analysis

  • ChIP-seq for studying histone modifications associated with FARSB regulation

  • Integration of multi-omics data using platforms like LinkedOmics

What are the latest approaches for constructing ceRNA networks involving FARSB?

Competing endogenous RNA (ceRNA) networks involving FARSB represent an emerging area of research:

• Methodological workflow for ceRNA network construction:

  • Target miRNA prediction: Using TargetScan, DIANA-microT, and RNAinter databases to identify miRNAs targeting FARSB

  • Target lncRNA identification: Using miRNet2.0 and starBase3.0 to predict lncRNAs that interact with identified miRNAs

  • Network construction: Building an lncRNA-miRNA-mRNA (FARSB) ceRNA network in HCC

  • Confirmation criteria: miRNAs appearing in three databases simultaneously with negative correlation to FARSB, and lncRNAs appearing in two databases with negative correlation to target miRNAs

• Validation approaches:

  • Luciferase reporter assays to confirm direct interactions

  • RNA pulldown and RIP assays to validate RNA-RNA or RNA-protein interactions

  • Expression correlation analysis using TCGA data

  • Functional studies to assess the biological significance of identified interactions

This network analysis helps understand the broader regulatory landscape affecting FARSB expression and function in disease contexts.

How might FARSB be targeted therapeutically in human diseases?

Therapeutic targeting of FARSB represents a promising research direction:

• Potential therapeutic strategies:

  • mTORC1 pathway modulation: Studies show rapamycin can reverse FARSB-mediated effects in HCC, with rapamycin treatment (4 mg/kg) significantly affecting tumor growth in xenograft models

  • Ferroptosis induction: Targeting FARSB to enhance sensitivity to ferroptosis inducers like erastin

  • Small molecule inhibitors: Developing specific inhibitors targeting FARSB function or interactions

• Experimental approaches for therapeutic validation:

  • Cell viability assays combining FARSB knockdown/overexpression with candidate therapeutics

  • In vivo preclinical models testing combined approaches

  • Investigation of synergistic effects with established therapies

• Monitoring therapeutic response:

  • ROS detection assays to measure oxidative stress changes

  • MDA detection for lipid peroxidation assessment

  • Imaging techniques to visualize and quantify response

How do evolutionary constraints affect FARSB function across species?

Understanding the evolutionary context of FARSB provides insights into its fundamental importance:

• Evolutionary conservation analysis:

  • FARSB belongs to aminoacyl-tRNA synthetases (ARSs), which are among the most conserved protein-coding genes in the human genome

  • Brain-related proteins show particularly strong conservation, suggesting tight functional constraints

• Methodological approaches:

  • Comparative genomics across species to identify conserved domains and variants

  • Positive selection analysis to detect regions under evolutionary pressure

  • Reconstruction of ancestral sequences to understand evolutionary trajectories

• Implications for disease research:

  • Highly conserved regions often correspond to functionally critical domains

  • Variants in evolutionarily constrained regions typically have higher pathogenicity

  • Cross-species models can provide insights into fundamental FARSB functions

What are the confounding factors in clinical interpretation of FARSB variants?

Accurate interpretation of FARSB variants presents several challenges:

• Variant classification considerations:

  • According to clinical studies, null variants in tRNA synthetase genes are typically lethal, suggesting viable patients must retain some residual activity

  • At least one variant in surviving patients with FARSB-related conditions must have residual activity

  • ACMG guidelines are used for pathogenicity classification of variants

• Genotype-phenotype correlation complexities:

  • FARSB mutations cause multisystem disorders with variable expressivity

  • Phenotypic overlap with other aminoacyl-tRNA synthetase-related diseases complicates diagnosis

  • Environmental and genetic modifiers may influence disease presentation

• Best practices for variant interpretation:

  • Multi-omics approach combining genomic, transcriptomic, and proteomic data

  • Functional validation through patient-derived cells or model systems

  • Population-specific frequency data consideration

  • Family segregation analysis

Whole Exome Sequencing has proven valuable for early diagnosis of atypical cases of FARSB mutation in children with multisystem disorders, enabling potential novel treatments and facilitating carrier detection and prenatal diagnosis .

How can single-cell technologies advance our understanding of FARSB function?

Single-cell approaches offer unprecedented resolution for studying FARSB:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveals cell-specific expression patterns of FARSB across tissues and in disease states

  • Identifies cell populations particularly sensitive to FARSB dysfunction

  • Enables trajectory analysis to understand disease progression at cellular level

• Single-cell proteomics:

  • Measures FARSB protein levels and modifications at single-cell resolution

  • Identifies cell-specific interactomes

• Spatial transcriptomics/proteomics:

  • Maps FARSB expression and function in tissue context

  • Reveals spatial relationships with other disease-relevant factors

These technologies could particularly benefit understanding of FARSB's role in complex tissues like brain and liver, where cellular heterogeneity is pronounced.

What is the relationship between FARSB and non-canonical cellular processes?

Beyond its canonical role in protein synthesis, FARSB may participate in non-canonical functions:

• Potential non-canonical functions:

  • Signaling pathway modulation (e.g., mTORC1 pathway, as demonstrated in HCC studies)

  • Regulation of cell death mechanisms (particularly ferroptosis)

  • Potential roles in cellular stress responses

• Investigative approaches:

  • Proximity labeling techniques (BioID, APEX) to identify novel interaction partners

  • Subcellular fractionation and localization studies

  • Stress-response assays under various cellular perturbations

• Disease relevance:

  • Non-canonical functions may explain tissue-specific manifestations of FARSB-related diseases

  • Could reveal novel therapeutic targets independent of protein synthesis

This research direction aligns with findings that aminoacyl-tRNA synthetases often have functions beyond their roles in protein synthesis, contributing to cellular signaling and metabolism .