Recombinant Mouse FAST kinase domain-containing protein 5 (Fastkd5)

What is the primary function of Fastkd5 in mitochondria?

Fastkd5 functions as an endonuclease responsible for processing non-canonical pre-mRNAs in the primary mitochondrial polycistronic transcript. Specifically, it cleaves pre-mRNAs that are not flanked by tRNAs, allowing for their maturation and subsequent translation. The complete loss of Fastkd5 function specifically affects the processing of only the non-canonical pre-mRNAs in the primary polycistronic transcript . This processing is essential for mitochondrial gene expression, as mRNAs with unprocessed 5'-UTRs generally cannot load efficiently onto mitoribosomes to form an initiation complex, preventing translation of mtDNA-encoded proteins .

Which mitochondrial transcripts are specifically processed by Fastkd5?

Fastkd5 is specifically responsible for processing the non-canonical pre-mRNAs in the mitochondrial transcript. In the absence of Fastkd5, there is a near-complete loss of the mature, processed forms of the CO1, CO3, and cytb mRNAs . These three transcripts are considered "non-canonical" because they are not flanked by tRNAs in the primary polycistronic transcript, unlike most other mitochondrial mRNAs which are processed through tRNA excision by RNase P and RNase Z . The inability to process these specific transcripts leads to translation defects primarily affecting COX I and cyt b proteins .

What are the essential domains of Fastkd5 protein?

Fastkd5 contains several functional domains that are all necessary for its proper function. These include:

• Mitochondrial targeting sequence (MTS) - directs the protein to mitochondria

• Heptatricopeptide repeats - likely involved in RNA binding/recognition

• FAST1 domain

• FAST2 domain

• RAP domain - contains an endonuclease-like fold

Deletion experiments have shown that removal of any of these domains (MTS, heptatricopeptide repeats, FAST1, FAST2, or RAP domains) results in loss of function . The heptatricopeptide repeat coiled-coil domains likely play a role in positioning RNA substrates for cleavage .

How can Fastkd5 knockout cell lines be generated?

Fastkd5 knockout cell lines can be generated using CRISPR/Cas9–mediated gene editing. The process involves:

• Designing a gene-specific target sequence (sgRNA)

• Cloning the sgRNA into a plasmid containing Cas9 and a selection marker (e.g., pSpCas9(BB)-2A-Puro)

• Transfecting the plasmid into target cells using a reagent like Lipofectamine 3000

• Selecting transfected cells with puromycin (typically 2.5 μg/ml for 2 days)

• Isolating single cell clones

• Screening clones for loss of Fastkd5 protein by immunoblotting

• Confirming frameshift mutations by genomic sequencing

Successful knockout cells should be maintained in medium containing pyruvate and uridine to support their growth, as they will likely have deficient OXPHOS activity .

What are the methods for expressing recombinant Fastkd5 protein?

Recombinant Fastkd5 protein can be expressed using several approaches:

• Retroviral expression in mammalian cells:

  • Clone Fastkd5 cDNA into a retroviral vector (e.g., pBABE-3xFLAG-Puro)

  • Transfect the construct into a packaging cell line (e.g., Phoenix cells)

  • Collect virus-containing medium after 48 hours

  • Infect target cells in the presence of polybrene (4 μg/ml)

• Insect cell expression system for protein purification:

  • Create a construct with Fastkd5 lacking the N-terminal mitochondrial targeting sequence (Δ1–27)

  • Clone into an appropriate vector with affinity tags (e.g., N-terminal 6xHis tag with TEV cleavage site)

  • Generate baculovirus

  • Express in insect cells (Sf9, Sf21, or Hi5 cells)

  • Purify using affinity chromatography

This approach is particularly useful for obtaining purified protein for in vitro assays.

How can Fastkd5 localization be verified in cells?

Fastkd5 localization can be verified through immunofluorescence experiments:

• Grow cells on coverslips for 24 hours

• Fix cells with 4% formaldehyde in PBS for 20 min at 37°C

• Wash with PBS and permeabilize with 0.5% Triton in PBS for 15 min

• Block with 5% BSA in PBS for at least 10 minutes

• Incubate with primary antibodies against Fastkd5 and mitochondrial markers

• Wash and incubate with secondary antibodies coupled with fluorochromes and DAPI

• Mount coverslips and image using confocal microscopy

This technique can confirm mitochondrial localization of wild-type Fastkd5 and can be used to assess the localization of mutant variants .

How can the RNA processing activity of Fastkd5 be assessed?

The RNA processing activity of Fastkd5 can be assessed through multiple approaches:

• Northern blot analysis:

  • Extract total RNA from wild-type and Fastkd5 knockout cells

  • Separate RNA by gel electrophoresis

  • Transfer to membrane and probe with specific probes for mitochondrial transcripts

  • Compare the presence of mature processed forms of non-canonical mRNAs (CO1, CO3, cytb)

• RT-qPCR analysis:

  • Design primers specific for processed and unprocessed forms of mitochondrial transcripts

  • Perform RT-qPCR to quantify relative levels of different transcript forms

• In vitro RNA processing assay:

  • Purify recombinant Fastkd5 protein

  • Synthesize RNA substrates with fluorescent tags (e.g., Cy3)

  • Incubate substrate with purified Fastkd5

  • Analyze cleavage products by gel electrophoresis

  • This approach can demonstrate the direct endonuclease activity of Fastkd5 on specific RNA substrates

How can the impact of Fastkd5 on mitochondrial translation be measured?

The impact of Fastkd5 on mitochondrial translation can be measured through pulse-labeling experiments:

• Incubate cells with [35S]-Met/Cys in the presence of emetine (an inhibitor of cytoplasmic translation)

• Extract total protein and separate by SDS-PAGE

• Detect newly synthesized mitochondrial proteins by autoradiography

• Quantify the relative synthesis rates of different mitochondrial proteins

In Fastkd5 knockout cells, this approach reveals specific decreases in the translation of COX I and cyt b, both encoded by non-canonical pre-mRNAs, consistent with impaired processing of these transcripts .

What cellular phenotypes are associated with Fastkd5 deficiency?

Fastkd5 deficiency leads to several observable cellular phenotypes:

• OXPHOS deficiency: Loss of Fastkd5 results in severe combined OXPHOS assembly defects due to the inability to translate mRNAs with unprocessed 5'-UTRs .

• Complex-specific defects: Immunoblot analysis shows specific decreases in COX I and cyt b proteins, while other mitochondrial proteins like ND1 or ATP6 remain unaffected .

• Growth defects: Fastkd5 knockout cells become uridine auxotrophs, requiring uridine supplementation for growth due to compromised OXPHOS capacity and its link to pyrimidine synthesis .

• Complete loss of Complex III and IV assembly: The translation defects lead to failure in assembling respiratory chain complexes III and IV, and because Complex I stability depends on Complex III, also results in loss of fully assembled Complex I .

These phenotypes can be assayed through growth assays, immunoblotting for OXPHOS components, and blue native PAGE for respiratory complex assembly.

How can the critical amino acid residues of Fastkd5 be identified?

Critical amino acid residues in Fastkd5 can be identified through a systematic mutagenesis approach:

• Selection of target residues:

  • Identify evolutionarily conserved residues across FASTKD5 proteins from different species

  • Include conserved residues in other FASTKD family members

  • Consider variants of unknown significance identified in patients

  • Focus on aspartate/glutamate residues that might participate in catalysis

• Mutagenesis and functional testing:

  • Generate point mutations (typically to alanine)

  • Express mutant proteins in Fastkd5 knockout cells

  • Assess rescue of function through:

    • Immunoblotting for restored COX I expression

    • Immunofluorescence with anti-COX I antibody

    • Northern blot analysis to examine pre-mRNA processing

    • Analysis of OXPHOS complex assembly

• Protein modeling:

  • Use structure prediction tools like AlphaFold to model FASTKD5 protein structure

  • Model individual point mutations using molecular visualization software

  • Correlate structural predictions with functional data

This approach has revealed that different residues are required for processing different RNA substrates, suggesting substrate-specific mechanisms .

What is known about substrate specificity of Fastkd5?

The substrate specificity of Fastkd5 shows several interesting characteristics:

• Substrate selection: Fastkd5 specifically processes non-canonical pre-mRNAs (CO1, CO3, and cytb) but not other mitochondrial transcripts .

• Substrate-specific requirements: Mutation analysis revealed that while many amino acid residues are required for processing pre-CO1, a smaller subset of these same essential residues are required for processing pre-CO3, and even fewer for pre-cytb .

• Recognition mechanism: Unlike tRNAs that have characteristic tertiary structures recognized by RNase P and RNase Z, there does not appear to be a common sequence or RNA structure that denotes processing sites for Fastkd5 .

• In vitro specificity: Purified Fastkd5 can process synthetic RNA substrates at the expected sites but does not process non-specific RNA sequences, confirming that the activity is not due to general nuclease activity .

• Additional specificity factors: The observation that additional cleavage products are present when processing authentic substrates in vitro suggests that other factors may be necessary for complete specificity or efficient cleavage in vivo .

How does Fastkd5 interact with other mitochondrial RNA processing factors?

The interaction of Fastkd5 with other mitochondrial RNA processing factors reveals complex relationships:

What are the best experimental controls for Fastkd5 studies?

When conducting Fastkd5 research, several important controls should be included:

• For knockout studies:

  • Multiple independent knockout clones to rule out off-target effects

  • Rescue experiments with wild-type Fastkd5 to confirm phenotype specificity

  • Parental cell line as positive control

• For protein expression studies:

  • Empty vector controls

  • Expression of unrelated mitochondrial proteins to control for overexpression effects

  • Tagged versions of Fastkd5 to verify expression levels

• For RNA processing assays:

  • Non-substrate RNAs to confirm specificity

  • Catalytically inactive Fastkd5 mutants

  • Time course experiments to assess processing kinetics

• For localization studies:

  • Co-staining with established mitochondrial markers

  • Controls for antibody specificity using knockout cells

How can recombinant Fastkd5 be optimized for in vitro studies?

Optimizing recombinant Fastkd5 for in vitro studies involves several considerations:

• Construct design:

  • Remove the mitochondrial targeting sequence (Δ1-27) to improve solubility

  • Include appropriate affinity tags (e.g., 6xHis tag) for purification

  • Include a protease cleavage site (e.g., TEV) to remove tags after purification

• Expression system:

  • Insect cell expression systems (Sf9, Sf21, or Hi5 cells) have been successfully used

  • Baculovirus-based expression provides good yields of functional protein

• Purification conditions:

  • Optimize buffer conditions to maintain protein stability and activity

  • Consider the addition of stabilizing agents if needed

  • Test different purification strategies to maximize yield and purity

• Activity verification:

  • Design RNA substrates with fluorescent tags for easy detection

  • Establish dose-dependency of activity

  • Verify substrate specificity with control RNAs

What are the challenges in studying Fastkd5-RNA interactions?

Studying Fastkd5-RNA interactions presents several challenges:

• Substrate identification:

  • No common sequence or RNA structure has been identified that denotes processing sites

  • Multiple cleavage sites may exist in a single substrate

  • Some substrates may require additional factors for optimal recognition

• Mechanistic analysis:

  • The exact catalytic mechanism remains undefined

  • Different substrates appear to have different requirements for processing

  • Distinguishing between RNA binding and catalytic activities

• Structural considerations:

  • The exact structure of Fastkd5-RNA complexes is not yet determined

  • The role of the heptatricopeptide repeats in RNA recognition needs further characterization

  • Potential conformational changes upon substrate binding

• Kinetic analysis:

  • Current assays are often endpoint measurements rather than kinetic

  • Establishing physiologically relevant reaction conditions

  • Comparing in vitro rates with estimated in vivo processing rates

How does Fastkd5 activity coordinate with mitochondrial transcription?

The coordination between Fastkd5 activity and mitochondrial transcription represents an important research direction:

• Co-transcriptional processing: The idea that RNA processing occurs co-transcriptionally implies that all primary processing events should have similar kinetics. Recent estimates indicate that the rate of transcription of the polycistronic transcript is <1kb/min and that the half-lives of the pre-mRNAs (canonical and non-canonical) are in the range of 1-39 min, consistent with coupling between transcription and processing .

• Processing order: Bhatta et al. showed that for tRNA processing, 5' processing by RNase P precedes 3' cleavage by RNase Z. The relationship between canonical tRNA processing and non-canonical processing by Fastkd5 needs further investigation .

• Factors affecting processing efficiency: Investigating how transcription rate, RNA secondary structure, and the assembly of processing complexes affect Fastkd5 activity could provide insights into the coordination of these processes.

• Spatial organization: Determining whether Fastkd5 is part of a larger mitochondrial RNA processing complex or "mitochondrial RNA granule" could reveal how different RNA processing activities are coordinated spatially within mitochondria.

What is the evolutionary significance of the Fastkd protein family?

Understanding the evolutionary aspects of the Fastkd protein family can provide insights into their specialized functions:

• Evolutionary conservation: Analysis of Fastkd5 conservation across species and comparison with other Fastkd family members can reveal functionally important regions and specializations.

• Functional divergence: Investigating how different Fastkd proteins (FASTKD1-5) have evolved specialized functions in mitochondrial RNA metabolism could highlight the importance of non-canonical RNA processing.

• Relationship to other RNA processing enzymes: Exploring potential evolutionary relationships between Fastkd proteins and other RNA processing enzymes might provide insights into the catalytic mechanism.

• Species-specific adaptations: Comparing mouse and human Fastkd5 activities could reveal species-specific adaptations in mitochondrial RNA processing pathways.

How might Fastkd5 dysfunction contribute to human disease?

The potential role of Fastkd5 in human disease is an important research direction:

• Mitochondrial disease: As Fastkd5 is essential for proper OXPHOS function through its role in mitochondrial RNA processing, dysfunction could contribute to mitochondrial disease phenotypes.

• Variants of unknown significance: Several variants of unknown significance in FASTKD5 have been identified in patients . Functional characterization of these variants could establish their pathogenicity.

• Tissue-specific effects: Investigating tissue-specific expression and requirements for Fastkd5 could explain why mutations might affect certain tissues more than others.

• Therapeutic approaches: Understanding the precise function of Fastkd5 could lead to targeted therapeutic approaches for patients with Fastkd5 mutations, such as gene therapy or compounds that might bypass the processing defect.