Recombinant Human FAST kinase domain-containing protein 5 (FASTKD5)

What is the primary function of FASTKD5 in mitochondrial RNA processing?

FASTKD5 is a crucial protein responsible for processing non-canonical mitochondrial pre-mRNAs that are not flanked by tRNAs in the primary polycistronic transcript. It functions as an endonuclease that specifically recognizes and cleaves client RNA substrates at appropriate sites. While RNase P and RNase Z process canonical transcripts flanked by tRNAs, FASTKD5 processes transcripts such as CO1, CO3, and cytb pre-mRNAs, enabling their translation into functional proteins essential for oxidative phosphorylation (OXPHOS) .

The processing activity of FASTKD5 is particularly critical because mRNAs with unprocessed 5'-UTRs cannot efficiently load onto mitoribosomes to form initiation complexes, preventing translation of essential OXPHOS components .

Which mitochondrial transcripts are specifically processed by FASTKD5?

FASTKD5 specifically processes non-canonical pre-mRNAs in the primary mitochondrial polycistronic transcript. Based on experimental evidence, the key transcripts processed by FASTKD5 include:

• CO1 (encoding COX I)

• ATP8/6+CO3 (specifically the processing of CO3 from this tricistronic transcript)

• ND5+cytb (specifically the processing of cytb from this bicistronic transcript)

Northern blot analyses of FASTKD5 knockout cells show near-complete loss of the mature, processed forms of these mRNAs, confirming FASTKD5's specificity for these substrates .

What are the main structural domains of FASTKD5 and how do they contribute to its function?

FASTKD5 contains several functional domains that are all essential for its activity:

• MTS (Mitochondrial Targeting Sequence) - Directs the protein to mitochondria

• Heptatricopeptide repeats - Likely involved in RNA binding/recognition

• FAST1 domain - Essential for function, specific role not fully characterized

• FAST2 domain - Essential for function, specific role not fully characterized

• RAP (RNA-binding domain abundant in Apicomplexans) domain - Contains an endonuclease-like fold that may harbor the active site

Deletion experiments demonstrate that removing any of these domains results in complete loss of FASTKD5 function. This suggests that all domains work in concert to achieve proper substrate recognition and processing .

What is the most effective method for generating FASTKD5 knockout cell lines for functional studies?

CRISPR/Cas9-mediated gene editing has proven highly effective for generating complete FASTKD5 knockout cell lines. The methodological approach involves:

• Design of gene-specific target sequence (sgRNA targeting FASTKD5)

• Cloning the sgRNA into a vector containing Cas9 and a puromycin resistance marker (e.g., pSpCas9(BB)-2A-Puro (PX459) V2.0)

• Transfection into target cells (e.g., 143B cells) using Lipofectamine 3000

• Selection of transfected cells with puromycin (2.5 μg/ml) for 2 days

• Isolation of single cell clones

• Screening clones for loss of FASTKD5 protein by immunoblotting

• Confirmation of frameshift mutations by genomic sequencing

It's advisable to select at least two independent knockout clones for subsequent analyses to ensure phenotype consistency. When culturing FASTKD5 knockout cells, supplementation with pyruvate and uridine is necessary to support the replication of cells with deficient OXPHOS activity .

How can recombinant FASTKD5 protein be efficiently produced for in vitro studies?

For high-yield production of functional recombinant FASTKD5 protein, an insect cell expression system is recommended. The protocol involves:

• Cloning the FASTKD5 sequence (lacking the N-terminal mitochondrial targeting sequence, Δ1-27) into an appropriate vector (e.g., 438-C vector) with an N-terminal 6xHis tag followed by a TEV cleavage site

• Generating baculovirus in Sf9 and Sf21 cells

• Expressing the protein in Hi5 insect cells for large-scale production

• Purifying the protein using standard methods for His-tagged proteins

This approach yields functional FASTKD5 protein capable of processing RNA substrates in vitro. The removal of the mitochondrial targeting sequence (first 27 amino acids) is crucial for efficient expression while maintaining the protein's enzymatic activity .

What phenotypic changes should be expected in FASTKD5 knockout cells?

FASTKD5 knockout cells exhibit several characteristic phenotypic changes:

• Protein level changes: Specific decreases in COX I and cytochrome b proteins, with minimal effects on other mtDNA-encoded proteins like ND1 or ATP6

• RNA processing defects: Near-complete loss of mature, processed forms of CO1, CO3, and cytb mRNAs

• Translation defects: Severely impaired translation of COX I and cytochrome b proteins, while translation of other mitochondrial proteins, including COX III, remains largely unaffected

• OXPHOS complex assembly: Complete failure to assemble Complexes III and IV, and due to the dependence of Complex I stability on Complex III assembly, a complete loss of fully assembled Complex I

• Metabolic requirements: Uridine auxotrophy, as the cells require external uridine due to impaired pyrimidine synthesis resulting from OXPHOS dysfunction

These phenotypes can be rescued by retroviral expression of wild-type FASTKD5, confirming their specificity to FASTKD5 loss .

How can the RNA processing activity of FASTKD5 be demonstrated in vitro?

To demonstrate FASTKD5's RNA processing activity in vitro, a reconstituted system using purified components is recommended:

• Purify recombinant FASTKD5 protein (lacking the MTS) from insect cells

• Synthesize single-stranded RNA substrates corresponding to the natural pre-mRNA targets (CO1, ATP8/6+CO3, and ND5+cytb)

• Label the RNA substrates at the 3' end with a fluorophore (e.g., Cy3) for detection

• Incubate the labeled RNA substrates with varying concentrations of purified FASTKD5

• Analyze the reaction products by gel electrophoresis to identify specific cleavage products

In such assays, authentic FASTKD5 processing will generate specific fragments (e.g., a 27 nt band for the tested substrates). The processing should be FASTKD5 dose-dependent and substrate-specific, showing no activity on non-relevant RNA sequences .

What approaches can be used to map functionally critical amino acid residues in FASTKD5?

A systematic mutagenesis approach combined with functional assays provides the most comprehensive mapping of critical residues:

• Sequence analysis: Identify evolutionarily conserved residues across FASTKD5 proteins from different species and in other FASTKD family members

• Site-directed mutagenesis: Generate a panel of single amino acid substitutions (preferably to alanine) in a FASTKD5 expression construct

• Functional complementation: Express each mutant in FASTKD5 knockout cells

• Phenotypic rescue assessment:

  • Assess COX I and cytochrome b expression by immunoblotting

  • Conduct immunofluorescence analysis with anti-COX I antibody

  • Perform Northern blot analysis to evaluate pre-mRNA processing for different substrates

• Protein stability analysis: Measure half-life of selected variants after inhibition of cytosolic translation with cycloheximide

This approach has revealed that different amino acid residues show substrate-specific importance, with some residues being critical for processing all substrates while others are substrate-specific. Additionally, some residues (e.g., E317) affect protein stability rather than catalytic activity .

How does FASTKD5 substrate specificity vary between different RNA targets?

FASTKD5 shows notable substrate-specific requirements for processing different non-canonical pre-mRNAs:

This hierarchy of residue dependency suggests that FASTKD5 processes different RNA substrates through distinct molecular interactions, rather than using a "one size fits all" mechanism. The substrate-specific nature of these critical residues also explains why some FASTKD5 variants may affect the processing of one transcript but not others .

What controls should be included when evaluating FASTKD5 knockout phenotypes?

When evaluating FASTKD5 knockout phenotypes, the following controls are essential:

• Parental wild-type cells: Include the original cell line (e.g., 143B cells) as a positive control

• Multiple independent knockout clones: Analyze at least two independently derived knockout clones to ensure phenotype consistency

• Rescue with wild-type FASTKD5: Express wild-type FASTKD5 in knockout cells to demonstrate phenotype reversibility

• Vector-only control: Include empty vector controls for rescue experiments

• Multi-level assessment:

  • Protein level (immunoblotting)

  • RNA processing (Northern blotting)

  • Translation analysis (pulse-labeling with [35S]-Met/Cys)

  • Single-cell analysis (immunofluorescence)

  • Complex assembly (Blue Native PAGE)

These comprehensive controls ensure that observed phenotypes are specifically attributed to FASTKD5 loss and not to off-target effects or clonal variations .

How do partial and complete FASTKD5 deficiency phenotypes differ?

The phenotypic consequences of partial versus complete FASTKD5 deficiency show important differences:

This difference suggests that even small amounts of FASTKD5 protein may be sufficient for processing some substrates (particularly ND5+cytb), while other substrates require higher FASTKD5 levels. This finding has important implications for interpreting partial loss-of-function models and patient-derived cells with hypomorphic mutations .

What are the limitations of in vitro reconstituted systems for studying FASTKD5 function?

The in vitro reconstituted system for studying FASTKD5 function has several limitations:

• Incomplete specificity: While FASTKD5 processes all three non-canonical substrates at the expected sites, it also produces additional smaller cleavage products, suggesting that the in vitro system lacks factors that ensure complete processing specificity

• Lack of quantitative kinetics: Current endpoint assays cannot determine enzyme kinetics or processivity

• Uncoupling from transcription: The in vitro system does not recapitulate the likely co-transcriptional nature of RNA processing in vivo

• Absence of potential cofactors: FASTKD5 may utilize additional factors in vivo that serve as platforms for RNA recognition or enhance processivity

• Artificial substrate presentation: The synthetic RNA substrates may not perfectly mimic the natural substrate conformation in the context of the primary polycistronic transcript

These limitations suggest that while the in vitro system demonstrates FASTKD5's intrinsic processing capability, additional factors likely contribute to the complete specificity observed in vivo .

How does FASTKD5 deficiency impact oxidative phosphorylation system assembly?

FASTKD5 deficiency has dramatic consequences for OXPHOS assembly through a cascade of effects:

• Direct effects on protein synthesis: The inability to process CO1 and cytb pre-mRNAs prevents translation of these core subunits of Complex IV and Complex III, respectively

• Complex IV (cytochrome c oxidase) assembly: Complete loss of COX I prevents assembly of functional Complex IV

• Complex III (cytochrome bc1 complex) assembly: Absence of cytochrome b prevents assembly of functional Complex III

• Secondary Complex I deficiency: Due to the interdependence of Complexes I and III, wherein the stability of Complex I depends on assembled Complex III, FASTKD5 knockout also causes complete loss of fully assembled Complex I

• Metabolic consequences: The combined OXPHOS deficiency results in cells becoming uridine auxotrophs, as pyrimidine synthesis requires functional OXPHOS

This hierarchical collapse of the OXPHOS system highlights the critical role of proper RNA processing in maintaining mitochondrial function and explains why FASTKD5 dysfunction could cause severe mitochondrial disease phenotypes .

What is the relationship between FASTKD5 and other mitochondrial RNA processing factors?

FASTKD5 functions as part of the complete machinery required for processing the primary mitochondrial transcript, working in concert with other RNA processing factors:

While RNase P and RNase Z utilize additional subunits (TRMT10C, SDR5C1) as RNA recognition platforms, it remains unknown whether FASTKD5 requires additional factors for complete specificity in vivo. The co-transcriptional nature of mitochondrial RNA processing suggests that all primary processing events likely have similar kinetics, with recent estimates indicating that the rate of transcription of the polycistronic transcript is <1kb/min and pre-mRNA half-lives range from 1-39 minutes .

What are the implications of FASTKD5 research for understanding mitochondrial disease?

Research on FASTKD5 has significant implications for mitochondrial disease research:

• Disease mechanism: FASTKD5 dysfunction represents a novel mechanism of mitochondrial disease through defective RNA processing rather than direct defects in OXPHOS subunits or assembly factors

• Clinical spectrum: The hierarchical nature of substrate-specific effects suggests that partial FASTKD5 deficiency might present with variable clinical phenotypes depending on which substrates are most affected

• Variants of unknown significance: The functional mapping of critical amino acid residues provides a framework for interpreting patient variants in FASTKD5

• Therapeutic potential: Understanding the specific molecular mechanisms of FASTKD5 processing could enable the development of targeted therapies for patients with FASTKD5 mutations

• Biomarker development: The substrate-specific effects on different non-canonical pre-mRNAs could serve as molecular signatures for identifying FASTKD5-related disorders

The identification of variants of unknown significance in FASTKD5 in mitochondrial disease patients, combined with the functional data from experimental models, provides an important foundation for clinical correlation and potential therapeutic interventions .

How can structure-function relationships of FASTKD5 be comprehensively investigated?

A multi-faceted approach to investigating FASTKD5 structure-function relationships should include:

• Computational structural analysis:

  • AlphaFold-predicted structures as a framework

  • Mapping of functional residues identified through mutagenesis

  • Molecular dynamics simulations to predict substrate interactions

• Biochemical approach:

  • Systematic mutagenesis of conserved residues

  • Domain swapping experiments with other FASTKD family members

  • Cross-linking studies to identify RNA-protein contact points

• Biophysical methods:

  • Direct measurement of RNA binding affinities for different substrates

  • Structural studies (X-ray crystallography or cryo-EM) of FASTKD5 alone and in complex with substrate RNAs

  • Single-molecule approaches to observe processing in real-time

• Functional validation:

  • Complementation assays in knockout cells

  • RNA processing assays with multiple substrate variants

  • Tracking of substrate fate in cells expressing mutant variants

This comprehensive approach would bridge the gap between structural predictions and functional observations, providing mechanistic insights into how FASTKD5 recognizes and processes its specific substrates .

What experimental approach would best elucidate the specific RNA recognition elements for FASTKD5?

To identify the specific RNA elements recognized by FASTKD5, a systematic approach combining the following methods would be most effective:

• Systematic mutagenesis of RNA substrates:

  • Create a library of synthetic RNA substrates with systematic mutations in the sequence surrounding the cleavage sites

  • Test processing efficiency in the in vitro reconstituted system

  • Map critical nucleotides required for recognition and processing

• CLIP-seq (Cross-linking immunoprecipitation followed by sequencing):

  • Perform CLIP-seq with FASTKD5 to identify direct RNA binding sites in vivo

  • Compare binding profiles across different substrates to identify common motifs

• RNA structure probing:

  • Use SHAPE-seq or similar methods to determine the secondary structure of substrate RNAs

  • Compare structures of efficiently processed versus poorly processed variants

• Compensatory mutation analysis:

  • Introduce mutations that alter potential recognition elements

  • Test whether compensatory mutations in FASTKD5 can restore processing

  • Use this to map specific RNA-protein interaction interfaces

Unlike tRNAs processed by RNase P and RNase Z, which have characteristic tertiary structures, no common sequence or structure has been identified in FASTKD5 substrates. This systematic approach would help identify the elusive recognition elements that determine FASTKD5 specificity .

What are the most promising approaches for identifying potential FASTKD5 cofactors?

The identification of potential FASTKD5 cofactors would benefit from the following approaches:

• Proximity-dependent biotin identification (BioID):

  • Express FASTKD5 fused to a promiscuous biotin ligase

  • Identify proteins in close proximity to FASTKD5 in living cells

  • Focus particularly on RNA-binding proteins that could serve as recognition platforms

• Co-immunoprecipitation coupled with mass spectrometry:

  • Pull down FASTKD5 complexes from mitochondrial extracts

  • Identify co-precipitating proteins by mass spectrometry

  • Validate interactions through reciprocal co-immunoprecipitation

• Genetic screens:

  • Perform CRISPR screens to identify synthetic lethal interactions with FASTKD5 partial deficiency

  • Look for genetic modifiers that enhance or suppress FASTKD5 mutant phenotypes

• Biochemical fractionation and activity reconstitution:

  • Fractionate mitochondrial extracts and identify fractions that enhance FASTKD5 specificity in vitro

  • Purify components that restore complete processing specificity

Given that RNase P and RNase Z utilize additional subunits (TRMT10C, SDR5C1) as RNA recognition platforms, and that FASTKD5 shows incomplete specificity in vitro, the identification of potential cofactors would significantly advance understanding of mitochondrial RNA processing mechanisms .

How might FASTKD5 function be integrated into a comprehensive model of mitochondrial gene expression regulation?

To integrate FASTKD5 into a comprehensive model of mitochondrial gene expression regulation, researchers should consider:

• Spatiotemporal coordination:

  • Investigate whether RNA processing by FASTKD5 occurs co-transcriptionally

  • Determine if processing is coordinated with ribosome loading and translation

  • Map the physical locations of FASTKD5 activity within mitochondria

• Regulatory networks:

  • Explore potential regulatory mechanisms controlling FASTKD5 activity

  • Investigate whether FASTKD5 function is modulated by cellular stress or metabolic state

  • Determine if post-translational modifications affect FASTKD5 activity

• System-level integration:

  • Develop mathematical models of mitochondrial gene expression incorporating transcription, processing, and translation rates

  • Test how perturbations in FASTKD5 function propagate through the system

  • Identify rate-limiting steps and potential regulatory nodes

• Evolutionary perspective:

  • Compare FASTKD5 function across species with different mitochondrial genome organizations

  • Identify conserved and divergent aspects of non-canonical RNA processing

This integrated approach would position FASTKD5 within the broader context of mitochondrial gene expression regulation and might reveal unexpected connections to other cellular processes .