Affinage

FASTKD5

FAST kinase domain-containing protein 5, mitochondrial · UniProt Q7L8L6

Length
764 aa
Mass
86.6 kDa
Annotated
2026-06-09
10 papers in source corpus 7 papers cited in narrative 8 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/6 claims corpus-supported (83%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

FASTKD5 is a mitochondrial endonuclease that executes the post-transcriptional processing of non-canonical junctions in the primary polycistronic mitochondrial transcript—the pre-mRNA boundaries that are not flanked by tRNAs and therefore cannot be liberated by the canonical tRNA-punctuation machinery (PMID:25683715). It resides in mitochondrial RNA granules, the sites of organellar RNA processing and ribosome biogenesis (PMID:25683715). Reconstitution with purified protein and synthetic RNA established FASTKD5 as the sole biochemical factor required to cleave its three clients—the CO1, CO3, and CYB pre-mRNAs—at correct sites, with a key active-site residue needed for catalysis but dispensable for substrate binding, separating recognition from cleavage (PMID:40637235). Loss of FASTKD5 produces the most severe non-canonical processing defect among FASTK family members, abolishing maturation of these transcripts, preventing their translation, and causing a profound complex IV (cytochrome c oxidase) assembly and OXPHOS deficiency (PMID:25683715, PMID:34748562). Bi-allelic loss-of-function variants in FASTKD5 cause a human mitochondrial disorder in which impaired COX subunit 1 translation, defective complex IV assembly, and reduced cytochrome c oxidase activity are rescued by wild-type but not mutant cDNA (PMID:40499538). The mitochondrial adaptor NLRX1 associates with FASTKD5 to modulate this processing activity in response to metabolic and infectious cues, a regulatory link exploited during HIV-1 infection to enhance OXPHOS (PMID:29932989, PMID:33767427).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2015 High

    Resolved how mitochondrial pre-mRNAs lacking tRNA punctuation marks are matured, identifying FASTKD5 as the factor required for processing non-canonical junctions and linking that step to respiratory chain biogenesis.

    Evidence siRNA silencing with RNA-seq, western blot, and complex IV assembly assays in human cells, plus MRG proteomics and fractionation

    PMID:25683715

    Open questions at the time
    • Did not establish whether FASTKD5 itself is the catalytic nuclease or a recruiting factor
    • Cleavage sites and substrate set not biochemically defined
  2. 2018 Medium

    Identified a regulatory input to FASTKD5 by showing NLRX1 associates with it in the matrix and negatively tunes processing of complex I and IV transcripts, connecting RNA processing to respiratory output.

    Evidence Co-immunoprecipitation, submitochondrial fractionation, transcript processing and complex activity assays

    PMID:29932989

    Open questions at the time
    • Lacks mutagenesis controls defining the interaction interface
    • Mechanism by which NLRX1 alters processing not resolved
  3. 2021 High

    Quantified FASTKD5's place in the FASTK family using genetic knockouts, showing it drives the most severe non-canonical processing defect and cooperates with FASTKD4 in non-coding RNA processing.

    Evidence CRISPR knockout cell lines with comprehensive mitochondrial transcriptomics, translation and OXPHOS assays

    PMID:34748562

    Open questions at the time
    • Did not demonstrate direct catalysis in vitro
    • Basis of FASTKD4/FASTKD5 cooperativity not mechanistically dissected
  4. 2021 Medium

    Placed the FASTKD5–NLRX1 axis in a physiological context, showing HIV-1 induces their association to boost mitochondrial respiratory component expression and OXPHOS to fuel viral replication.

    Evidence Quantitative proteomics, co-IP, and metabolic assays in HIV-1-infected CD4+ T cells and humanized mice

    PMID:33767427

    Open questions at the time
    • FASTKD5's specific contribution to the phenotype is indirect
    • How infection triggers the association is unknown
  5. 2024 High

    Provided the first reconstitution evidence that purified FASTKD5 alone cleaves client mitochondrial RNAs at correct sites, establishing intrinsic endonuclease activity.

    Evidence In vitro reconstitution with purified protein, synthetic RNA cleavage assays, and mutagenesis scanning (preprint, later published as PMID 40637235)

    PMID:bio_10.1101_2024.07.18.603998

    Open questions at the time
    • Preprint stage at the time
    • Structural recognition mechanism not yet defined
  6. 2025 High

    Established FASTKD5 as the bona fide endonuclease for its three clients, mapping cleavage sites, identifying upstream structural recognition features, and separating catalysis from RNA binding via active-site mutagenesis.

    Evidence CRISPR KO, active-site/domain mutagenesis, reconstituted in vitro cleavage with purified protein and synthetic substrates, RNA structural analysis, and complementation

    PMID:40637235

    Open questions at the time
    • No high-resolution structure of the enzyme–RNA complex
    • Exact recognition code for the upstream structural elements not defined
  7. 2025 High

    Demonstrated human disease relevance by showing bi-allelic loss-of-function FASTKD5 variants cause complex IV deficiency, with full rescue by wild-type cDNA confirming pathogenicity and a protein-stability mechanism for one allele.

    Evidence Exome sequencing, immunoblot, translation, complex IV assembly and activity assays, and cDNA rescue in patient fibroblasts

    PMID:40499538

    Open questions at the time
    • Clinical spectrum and genotype–phenotype correlation not detailed in the corpus
    • Whether NLRX1 regulation is altered in patients unknown

Open questions

Synthesis pass · forward-looking unresolved questions
  • How FASTKD5 recognizes its specific cleavage sites at the molecular level and how NLRX1 binding mechanically gates catalysis remain unresolved.
  • No structure of FASTKD5 bound to substrate RNA
  • Mechanism coupling NLRX1 association to processing modulation undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140098 catalytic activity, acting on RNA 4 GO:0003723 RNA binding 2 GO:0016787 hydrolase activity 2
Localization
GO:0005739 mitochondrion 2
Pathway
R-HSA-8953854 Metabolism of RNA 3 R-HSA-1430728 Metabolism 2
Partners
FASTKD4NLRX1
Complex memberships
mitochondrial RNA granule

Evidence

Reading pass · 8 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2015 FASTKD5 is required for maturing precursor mRNAs not flanked by tRNAs (non-canonical junctions), a processing step distinct from the canonical tRNA punctuation pathway; silencing FASTKD5 rendered mature COX I mRNA almost undetectable, severely reducing COX I synthesis and causing a complex IV assembly defect. siRNA silencing followed by RNA-seq, western blot, and complex IV assembly assays in human cell lines Cell Reports High 25683715
2015 FASTKD5 localizes to mitochondrial RNA granules (MRGs), organellar structures dedicated to post-transcriptional RNA processing and ribosome biogenesis. Proteomics of isolated mitochondrial RNA granules; co-localization and fractionation experiments Cell Reports High 25683715
2018 NLRX1 associates with FASTKD5 in the mitochondrial matrix, and this association negatively regulates post-transcriptional processing of mitochondrial transcripts for key components of respiratory complex I and IV, modulating their activity and supercomplex formation. Co-immunoprecipitation, submitochondrial fractionation, mitochondrial transcript processing assays, complex activity measurements Biochimica et Biophysica Acta - Molecular Cell Research Medium 29932989
2021 Loss of FASTKD5 (single or combined knockout with FASTKD4) causes the most severe defect in mitochondrial non-canonical junction RNA processing among FASTK family members, leading to marked deficiencies in translation of key electron transport chain components and in oxidative phosphorylation; combined FASTKD4/FASTKD5 knockout revealed cooperative roles in non-coding RNA processing. CRISPR knockout cell lines, comprehensive mitochondrial transcriptome analysis (RNA-seq), translation assays, OXPHOS measurements PLoS Genetics High 34748562
2021 HIV-1 infection induces the association of NLRX1 with mitochondrial FASTKD5 to promote expression of mitochondrial respiratory complex components, enhancing OXPHOS to fuel viral replication. Quantitative proteomics, co-immunoprecipitation, metabolic assays in HIV-1-infected CD4+ T cells and humanized mice Nature Immunology Medium 33767427
2025 FASTKD5 is an endonuclease that directly cleaves three non-canonical mitochondrial pre-mRNAs (CO1, CO3, and CYB) at specific sites; structural features 13–15 nt upstream of the CO1 and CYB cleavage sites suggest recognition mechanisms; a key putative active-site residue was required for processing all three substrates but its mutation did not alter RNA substrate binding, separating catalysis from binding; purified FASTKD5 cleaved client substrates correctly in a reconstituted in vitro system, establishing it as the sole biochemical factor needed for non-canonical junction processing. CRISPR knockout cell lines, active-site and domain mutagenesis, reconstituted in vitro cleavage assay with purified protein and synthetic RNA substrates, RNA structural analysis, complementation assays Nucleic Acids Research High 40637235
2025 Bi-allelic loss-of-function variants in FASTKD5 in human patient fibroblasts cause reduced FASTKD5 protein levels, impaired translation of COX subunit 1, defective complex IV assembly, and decreased cytochrome c oxidase enzymatic activity; wild-type FASTKD5 cDNA rescued all defects while missense variant cDNAs did not, confirming pathogenicity and demonstrating that protein stability is the mechanism for one hypomorphic allele. Exome sequencing, immunoblot, translation assays, complex IV assembly assays, enzymatic activity assays, cDNA rescue experiments in patient fibroblasts American Journal of Human Genetics High 40499538
2024 Reconstituted in vitro system with purified FASTKD5 protein and synthetic RNA substrates confirmed that FASTKD5 alone can cleave client mitochondrial RNA substrates at correct sites but not non-specific sequences; amino acid residue mapping showed RNA substrate-specific requirements, arguing against a single universal active-site model. In vitro reconstitution with purified protein, synthetic RNA substrate cleavage assays, mutagenesis scanning bioRxiv (preprint)preprint High bio_10.1101_2024.07.18.603998

Source papers

Stage 0 corpus · 10 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2015 Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis. Cell reports 241 25683715
2021 Multi-omics analyses reveal that HIV-1 alters CD4+ T cell immunometabolism to fuel virus replication. Nature immunology 96 33767427
2020 Nuclear-encoded mitochondrial ribosomal proteins are required to initiate gastrulation. Development (Cambridge, England) 35 32376682
2018 NLRX1 resides in mitochondrial RNA granules and regulates mitochondrial RNA processing and bioenergetic adaptation. Biochimica et biophysica acta. Molecular cell research 35 29932989
2021 The FASTK family proteins fine-tune mitochondrial RNA processing. PLoS genetics 32 34748562
2024 Analysis of 206 whole-genome resequencing reveals selection signatures associated with breed-specific traits in Hu sheep. Evolutionary applications 19 38911262
2021 Systematic Analysis of FASTK Gene Family Alterations in Cancer. International journal of molecular sciences 11 34768773
2025 FASTKD5 processes mitochondrial pre-mRNAs at noncanonical cleavage sites. Nucleic acids research 5 40637235
2022 Identification of human mitochondrial RNA cleavage sites and candidate RNA processing factors. BMC biology 5 35869520
2025 Bi-allelic mutations in FASTKD5 are associated with cytochrome c oxidase deficiency and early- to late-onset Leigh syndrome. American journal of human genetics 1 40499538

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