CDK5RAP1 is a mitochondrial radical S-adenosyl-L-methionine (SAM) methylthiotransferase of the MiaB subfamily. It converts N6-isopentenyladenosine at position 37 (i6A37) into 2-methylthio-N6-isopentenyladenosine (ms2i6A37) in four mitochondrial DNA-encoded tRNAs (Ser(UCN), Phe, Tyr and Trp). It uses two [4Fe-4S] clusters: one bound by the MTTase N-terminal domain, and a radical SAM cluster that cleaves SAM. The protein has an N-terminal mitochondrial transit peptide, an MTTase N-terminal domain, a radical SAM core and a C-terminal TRAM domain, and acts in the mitochondrial matrix. The ms2 modification stabilizes codon-anticodon interaction and is needed for efficient and accurate mitochondrial protein synthesis. Cdk5rap1-deficient mice have respiratory defects and stress-induced myopathy and cardiac dysfunction, and ms2 levels are reduced in patients with mitochondrial disease. Splice variants lacking the transit peptide can be found in the cytoplasm and nucleus, but ms2i6A is restricted to mitochondrial tRNA. The protein was first identified as a binding partner of the CDK5 activator p35 (CDK5R1) that inhibits CDK5 activation in vitro, which is the origin of its name. The physiological relevance of this interaction is unclear.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM signatures to the root catalytic activity term. Reason: True but uninformative. Per the RADICAL_SAM treatment rule, replaced by the specific reaction: i6A37 to ms2i6A37 tRNA methylthiotransferase (EC 2.8.4.3, GO:0035597), which the gene already carries by IMP and IBA. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0005654 nucleoplasm | IDA PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | MARK AS OVER ANNOTATED | Summary: Reiter et al. 2012 detected an MTS-less CDK5RAP1 variant and immunostained CDK5RAP1 in the nucleus, and proposed that it modifies nuclear RNA. Reason: The extra-mitochondrial signal comes from CDK5RAP1 variant 2, which lacks the mitochondrial targeting sequence and was overexpressed, together with antibody staining. The activity this location implies (ms2i6A in nuclear RNA) was later refuted: ms2i6A is absent from rho0 cells, which lack mitochondrial tRNAs, and the ms2i6A signal co-localizes with mitochondria (PMID:28981754). The MTS-less variant also lacks a conserved cysteine of the MTTase N-terminal domain. The protein may be detectable outside mitochondria, but saying it is active there overstates the evidence. Supporting Evidence: PMID:22422838 This distribution was confirmed by immunocytochemical (ICC) staining of CDK5RAP1, however with a stronger preference for nuclear localization (Figure 1c). PMID:28981754 Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species. PMID:28981754 However, the cytosolic CDK5RAP1_v2 not only lacks mitochondria-targeting signal, but also lacks an important cysteine residue in the UPF0004 domain (10). |
| GO:0005739 mitochondrion | EXP PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | ACCEPT | Summary: GFP-tagged CDK5RAP1 variant 1, which has the predicted mitochondrial import signal, co-localized with MitoTracker. Reason: Core location. The canonical isoform has an N-terminal mitochondrial transit peptide, and its substrates are the four mtDNA-encoded tRNAs (PMID:25738458). Supporting Evidence: PMID:22422838 The confocal analysis clearly shows a mitochondrial localization of variant 1. file:human/CDK5RAP1/CDK5RAP1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:22422838}. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: Detected in the MitoCoP high-confidence human mitochondrial proteome. Reason: High-throughput support that agrees with the targeted localization (PMID:22422838) and with the mitochondrial tRNA substrates. Supporting Evidence: PMID:34800366 mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). PMID:22422838 The confocal analysis clearly shows a mitochondrial localization of variant 1. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of mitochondrial localization for the vertebrate CDK5RAP1 clade (node PTN000228777), with mouse Cdk5rap1 and human CDK5RAP1 as sources. Reason: Agrees with the experimental localization and the mitochondrial tRNA substrates; the node placement is sound for the MTS-bearing animal clade. Supporting Evidence: PMID:22422838 The confocal analysis clearly shows a mitochondrial localization of variant 1. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: Human Protein Atlas immunofluorescence curation: mitochondrial staining. Reason: Independent antibody-based support for mitochondrial localization; agrees with PMID:22422838 and PMID:28981754. Supporting Evidence: file:human/CDK5RAP1/CDK5RAP1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:22422838}. PMID:28981754 The spotty signals stained by the anti-ms2i6A antibody were nicely co-localized with Mitotracker in HeLa cells but had disappeared from HeLa Ο0 cells (Figure 4A). |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (Compara from mouse Q8BTW8, UniProt SubCell SL-0173). Reason: Agrees with the experimental localization. Supporting Evidence: file:human/CDK5RAP1/CDK5RAP1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:22422838}. |
| GO:0005759 mitochondrial matrix | IDA PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | ACCEPT | Summary: Mitochondrial matrix assigned from the mitochondrial localization of variant 1 in Reiter et al. 2012. Reason: The paper shows mitochondrial co-localization rather than sub-compartment fractionation. Matrix is still the right site of action: the substrates, mtDNA-encoded tRNAs, are in the matrix, and the ms2i6A signal co-localizes with the matrix translation factor EF-Tu (PMID:28981754). Accepted as the most specific location. Supporting Evidence: PMID:22422838 The mitochondrial localization of CDK5RAP1 variant 1 is in full agreement with the suggested MiaB-like function in ms2i6A biosynthesis in mitochondrial tRNA. PMID:28981754 Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms2i6A in mitochondria and co-localization with the mitochondrial elongation factor Tu. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT inference of cytosolic localization from the deep MiaB node (PTN000228824), seeded by E. coli MiaB (P0AEI1), which is cytosolic because bacteria lack organelles. Reason: Cytosol is the ancestral compartment for bacterial MiaB, but the vertebrate CDK5RAP1 clade gained a mitochondrial transit peptide and acts on mitochondrial tRNAs (PMID:25738458, PMID:28981754). The cytosolic signal for human CDK5RAP1 comes from an MTS-less variant whose activity outside mitochondria was refuted. The deep-node assertion does not fit this lineage. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000228824 SUPPORTS SOURCE BUT NOT TARGET Deep MiaB node; cytosol is right for bacterial MiaB (E. coli P0AEI1), but the vertebrate CDK5RAP1 clade is mitochondrially targeted. UniProtKB:P0AEI1 SUPPORTS SOURCE BUT NOT TARGET E. coli MiaB is cytosolic because bacteria have no organelles; this location does not transfer to a mitochondrial paralog. UniProtKB:Q96SZ6 SOURCE WEAK OR INFERRED The human cytosol IDA comes from an overexpressed MTS-less variant whose extra-mitochondrial activity was refuted (PMID:28981754). Supporting Evidence: PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. PMID:28981754 Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species. file:human/CDK5RAP1/CDK5RAP1-uniprot.txt Absence of the mitochondrial target sequence which may lead to miss-localization. |
| GO:0005829 cytosol | IDA PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | MARK AS OVER ANNOTATED | Summary: Reiter et al. 2012 found overexpressed CDK5RAP1 variant 2, which lacks the mitochondrial import signal, mainly in the cytoplasm. Reason: The extra-mitochondrial signal comes from CDK5RAP1 variant 2, which lacks the mitochondrial targeting sequence and was overexpressed, together with antibody staining. The activity this location implies (ms2i6A in nuclear RNA) was later refuted: ms2i6A is absent from rho0 cells, which lack mitochondrial tRNAs, and the ms2i6A signal co-localizes with mitochondria (PMID:28981754). The MTS-less variant also lacks a conserved cysteine of the MTTase N-terminal domain. The protein may be detectable outside mitochondria, but saying it is active there overstates the evidence. Supporting Evidence: PMID:22422838 Variant 2, which is lacking the putative import signal, we found distributed in both cytoplasm and nucleus, respectively (Figure 1b), with the stronger signal appearing in the cytoplasm. PMID:28981754 Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species. PMID:28981754 However, the cytosolic CDK5RAP1_v2 not only lacks mitochondria-targeting signal, but also lacks an important cysteine residue in the UPF0004 domain (10). |
| GO:0006400 tRNA modification | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the MTTase/MiaB signatures to tRNA modification. Reason: True but general. CDK5RAP1 performs one specific tRNA modification, 2-methylthiolation of i6A37 in mitochondrial tRNAs, so the process is refined to tRNA methylthiolation (GO:0035600) and mitochondrial tRNA modification (GO:0070900). The gene carries both already. Proposed replacements: tRNA methylthiolation mitochondrial tRNA modification Supporting Evidence: PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. PMID:22422838 Transfection with esiRNA resulted indeed in a 78% decrease of ms2i6A content after 72 h in comparison to cells treated with esiRNA against green fluorescent protein (Figure 4b). |
| GO:0007420 brain development | NAS PMID:10915792 Identification of a common protein association region in the... | REMOVE | Summary: Author-statement annotation from a paper mapping the common binding region of three p35 (Nck5a)-binding proteins, including C42/CDK5RAP1. Reason: The paper maps where CDK5RAP1 binds p35 in vitro and contains no experiment on brain development. The process is the developmental role of CDK5/p35, transferred to a binding partner. No CDK5RAP1 loss-of-function study reports a brain-development phenotype; the knockout phenotype is mitochondrial translation failure with myopathy (PMID:25738458). The NAS has no supporting data and is not biologically plausible for a mitochondrial tRNA-modifying enzyme. Supporting Evidence: PMID:10915792 The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. PMID:25738458 Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. |
| GO:0009451 RNA modification | IMP PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | MODIFY | Summary: esiRNA knockdown of CDK5RAP1 in HeLa cells reduced ms2i6A by 78%. Reason: The knockdown evidence is sound, but RNA modification is too general. The modification is 2-methylthiolation of i6A37 in tRNA. The proposed nuclear (non-tRNA) RNA substrates were later refuted (PMID:28981754), so the specific supported processes are tRNA methylthiolation and mitochondrial tRNA modification. Proposed replacements: tRNA methylthiolation mitochondrial tRNA modification Supporting Evidence: PMID:22422838 Transfection with esiRNA resulted indeed in a 78% decrease of ms2i6A content after 72 h in comparison to cells treated with esiRNA against green fluorescent protein (Figure 4b). PMID:28981754 Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species. |
| GO:0016740 transferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the MTTase signatures to transferase activity. Reason: True but general; refined to the specific activity GO:0035597, which the gene already carries. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0019901 protein kinase binding | NAS PMID:10721722 Cloning of three novel neuronal Cdk5 activator binding prote... | KEEP AS NON CORE | Summary: Ching et al. 2000 isolated C42 (CDK5RAP1) as a p35-binding protein in a yeast two-hybrid screen; GST-C42 co-precipitated p35 complexed with Cdk5 from insect-cell lysate. Reason: The interaction is with the Cdk5/p35 kinase complex, through the activator p35, and was shown only in vitro and in heterologous systems. Its physiological relevance is doubtful: mature CDK5RAP1 is in the mitochondrial matrix and p35 is not. Kept as non-core, recording the historical CDK5 link, because the full text was not available. Supporting Evidence: PMID:10721722 The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. |
| GO:0035596 methylthiotransferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the MTTase N-terminal domain to methylthiotransferase activity. Reason: True but general. CDK5RAP1 is a MiaB-subfamily MTTase acting on i6A37 and not on t6A37 (PMID:22422838), so the activity is refined to GO:0035597. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: file:human/CDK5RAP1/CDK5RAP1-uniprot.txt Belongs to the methylthiotransferase family. MiaB subfamily. PMID:22422838 Again, the content of ms2t6A is not affected by knockdown of CDK5RAP1, proving that t6A is not recognized as a substrate of CDK5RAP1 |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of ms2i6A37 synthase activity for the MiaB clade (PTN000228824), with E. coli MiaB, B. subtilis and Thermotoga MiaB homologs and human CDK5RAP1 as experimental sources. Reason: Core function. The MiaB activity is ancestral to this clade, and human CDK5RAP1 has its own experimental support (E. coli complementation and knockdown, PMID:22422838; knockout mice, PMID:25738458). The target appearing in its own WITH/FROM reflects that experimental grounding. Supporting Evidence: PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (Compara from mouse Q8BTW8, RHEA:37067, EC 2.8.4.3). Reason: Agrees with the experimental evidence. Supporting Evidence: PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IMP PMID:22422838 The CDK5 repressor CDK5RAP1 is a methylthiotransferase actin... | ACCEPT | Summary: Knockdown of CDK5RAP1 reduced ms2i6A without affecting ms2t6A, and the MTS-less variant restored ms2i6A in miaB-deficient E. coli. Reason: Core function; directly supported. Mouse knockout data confirm the activity on mt-tRNA Ser(UCN), Phe, Tyr and Trp (PMID:25738458). Supporting Evidence: PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. PMID:22422838 Transfection with esiRNA resulted indeed in a 78% decrease of ms2i6A content after 72 h in comparison to cells treated with esiRNA against green fluorescent protein (Figure 4b). PMID:22422838 Again, the content of ms2t6A is not affected by knockdown of CDK5RAP1, proving that t6A is not recognized as a substrate of CDK5RAP1 |
| GO:0035600 tRNA methylthiolation | IEA GO_REF:0000108 | ACCEPT | Summary: Inferred from GO:0035597 by inter-ontology link. Reason: Core process: CDK5RAP1 itself catalyses the methylthiolation step converting i6A37 to ms2i6A37. Supporting Evidence: PMID:22422838 Transfection with esiRNA resulted indeed in a 78% decrease of ms2i6A content after 72 h in comparison to cells treated with esiRNA against green fluorescent protein (Figure 4b). PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0045664 regulation of neuron differentiation | NAS PMID:10721722 Cloning of three novel neuronal Cdk5 activator binding prote... | REMOVE | Summary: Author-statement annotation from the yeast two-hybrid identification of p35-binding proteins, based on the neuronal role of Cdk5/p35. Reason: The abstract attributes neuronal differentiation to Cdk5/p35 (Nclk), not to C42/CDK5RAP1, and the paper tests only binding and tissue expression (expression is broad, not neuron-restricted). This is guilt by association with a binding partner. No later study supports a role for CDK5RAP1 in neuron differentiation; the established function is mitochondrial tRNA modification (PMID:25738458). Supporting Evidence: PMID:10721722 Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. |
| GO:0045736 negative regulation of cyclin-dependent protein serine/threonine kinase activity | IDA PMID:11882646 Identification of a neuronal Cdk5 activator-binding protein ... | KEEP AS NON CORE | Summary: Ching et al. 2002 report that C42 (CDK5RAP1) specifically inhibits activation of Cdk5 by the activator Nck5a/p35 and co-immunoprecipitates with p35 in cultured cells. Reason: Experimental annotation whose full text is not in the cache, so it is not overruled. Biologically the claim is peripheral at best. The inhibition was shown in vitro or with overexpression; mature CDK5RAP1 is targeted to the mitochondrial matrix; and its paralog CDKAL1 does neither (PMID:21841312). No knockout phenotype attributable to CDK5 activation has been reported. Kept as non-core. Supporting Evidence: PMID:11882646 Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. |
| GO:0045786 negative regulation of cell cycle | TAS PMID:17491008 Microtubule association of the neuronal p35 activator of Cdk... | UNDECIDED | Summary: Traceable author statement from a paper on p35 binding to microtubules. Reason: The cached abstract is about p35-microtubule interactions and does not mention CDK5RAP1/C42, and the full text is unavailable, so the statement behind this TAS cannot be found. CDK5 is not a cell-cycle CDK, and nothing in the CDK5RAP1 literature reviewed here shows a cell-cycle role. Left undecided rather than overruled. Supporting Evidence: PMID:17491008 p35 binds directly to alpha/beta-tubulin and microtubules. |
| GO:0051246 regulation of protein metabolic process | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA machine-learning rule assigning regulation of protein metabolic process. Reason: CDK5RAP1 does not regulate protein metabolism as such. It installs a tRNA modification needed for efficient and accurate mitochondrial translation (PMID:25738458). The ARBA term is a loose proxy for that downstream effect, and the specific catalytic process is captured by GO:0035600 and GO:0070900. Supporting Evidence: PMID:25738458 Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM signatures to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to GO:0051539 4 iron, 4 sulfur cluster binding. CDK5RAP1 has the two [4Fe-4S] cluster-binding cysteine sets of MiaB enzymes, and the gene already carries GO:0051539. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:human/CDK5RAP1/CDK5RAP1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from MTTase and radical SAM signatures to 4Fe-4S cluster binding. Reason: Core cofactor binding per the RADICAL_SAM treatment rule. The MTTase N-terminal cluster (Cys109, Cys145, Cys183) and the radical SAM cluster (Cys258, Cys262, Cys265) are both conserved. The activity is established (PMID:22422838), so catalysis, and hence cluster use, is not in doubt. Supporting Evidence: file:human/CDK5RAP1/CDK5RAP1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:22422838 we detected significantly elevated levels of ms2i6A in the complemented cells (Figure 4a), showing that CDK5RAP1 has indeed MiaB-like catalytic properties. |
| GO:0070900 mitochondrial tRNA modification | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of mitochondrial tRNA modification for the vertebrate CDK5RAP1 clade (PTN000228777), with mouse Cdk5rap1 as a source. Reason: Core process. CDK5RAP1 modifies A37 of four mtDNA-encoded tRNAs (PMID:25738458), and ms2i6A is specific to mitochondrial tRNA (PMID:28981754). Supporting Evidence: PMID:25738458 In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. PMID:28981754 Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species. |
| GO:1904047 S-adenosyl-L-methionine binding | ISS PMID:15339930 MiaB protein is a bifunctional radical-S-adenosylmethionine ... | NEW | Summary: CDK5RAP1 is a MiaB-subfamily radical SAM methylthiotransferase. SAM binds the unique iron of the radical SAM [4Fe-4S] cluster and is cleaved to the 5'-deoxyadenosyl radical; a second SAM supplies the methyl group. Reason: Not in GOA. Added for consistency with the RADICAL_SAM treatment rule (SAM binding is a core cosubstrate-binding function of every active radical SAM enzyme) and with the LIAS and ELP3 reviews. This is a molecular function, so CDK5RAP1 itself must bind SAM, and it does: its reaction (RHEA:37067) consumes two SAM, and UniProt (PROSITE-ProRule PRU00780) records an exchangeable SAM on the radical SAM cluster. The bacterial ortholog MiaB, reconstituted in vitro, cleaves one AdoMet to 5'-deoxyadenosine and uses a second as the methyl donor (PMID:15339930). Transferred by sequence similarity (ISS) from MiaB. Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. file:human/CDK5RAP1/CDK5RAP1-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
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Download this section (compressed HTML)Q: Is the reported p35 (CDK5R1) binding and in vitro inhibition of CDK5 activation by CDK5RAP1 physiologically relevant, given that the mature enzyme is in the mitochondrial matrix? If so, which isoform does it, and where in the cell?
Q: Do the MTS-less splice variants (isoforms 2, 5, 6) have any catalytic activity or a non-catalytic role, given that they lack a conserved MTTase-domain cysteine?
Experiment: In vitro reconstitution of purified mature human CDK5RAP1 with [4Fe-4S] clusters and i6A37-containing mitochondrial tRNA transcripts, with SAM and reductant, to measure kinetics, tRNA selectivity (Ser(UCN), Phe, Tyr and Trp versus other i6A tRNAs) and the sulfur source.
Experiment: Test whether CDK5RAP1 loss alters CDK5 activity in neurons by comparing CDK5 kinase activity and p35 complexes in Cdk5rap1-knockout and wild-type brain, and by rescuing with MTS-intact and MTS-less variants.
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