mss116 encodes a nuclear-encoded, mitochondrially targeted DEAD-box ATP-dependent RNA helicase of fission yeast, one of four mitochondrial DEAD-box proteins in this organism (with Mrh5, Rpm2 and Irc3). It carries an N-terminal mitochondrial transit peptide, a Q motif, and the helicase ATP-binding and C-terminal domains typical of the family. The protein is inside mitochondria, protected from external protease and largely soluble, with a minor membrane-associated pool. Deletion of mss116 abolishes respiratory growth, causes loss of viability in stationary phase and strongly reduces synthesis of all mitochondrially encoded proteins, whether or not the mitochondrial genome carries introns. Mitochondrial ribosomal proteins of both subunits are destabilised and fully assembled mitoribosomes are reduced, while mitochondrial rRNA levels are unchanged. The protein itself does not co-sediment with mitoribosomes or their subunits. In cells with intron-containing mtDNA, mature cox1 and cob1 mRNAs are lost and intron-retaining precursors accumulate; removing the introns restores the mRNAs but not translation. Because splicing of these introns depends on intron-encoded maturases that are themselves made by mitochondrial translation, it is unresolved whether mss116 acts on the introns directly, as its budding-yeast ortholog does as an RNA chaperone for group I and II intron splicing. The budding-yeast protein cannot substitute for mss116, and the cox1-specific translational role of the budding-yeast ortholog appears to be carried out in fission yeast by the Mrh5 complex.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the DEAD/DEAH-box helicase domain. Correct but uninformative; the substrates of the characterised ortholog are RNAs (mitochondrial introns, rRNA, mRNA 5'-UTRs), and the fission-yeast authors propose mt-rRNA as the target of mss116. Reason: The term is true but less specific than the evidence supports. The DEAD-box family and the budding-yeast ortholog act on RNA, and the gene already carries RNA helicase activity, so RNA binding is the appropriate refinement. Proposed replacements: RNA binding Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt Belongs to the DEAD box helicase family. DDX18/HAS1 PMID:15618406 splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones PMID:38604460 we suggest that spMss116 may play a role in mitoribosome biogenesis via targeting of mt-rRNA(s). |
| GO:0003724 RNA helicase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of RNA helicase activity from a deep DEAD-box node whose seeds include S. cerevisiae MSS116 (SGD:S000002602) itself. mss116 retains all nine conserved DEAD-box motifs, including the Q motif, the ATP-binding P-loop and the DEAD box, so there is no sign of loss of catalytic function. Reason: The conserved catalytic motifs are intact and the closest characterised ortholog requires its ATP-dependent helicase activity for function in vivo. No fission-yeast biochemistry or catalytic-mutant analysis exists, but nothing argues against inheritance from the node. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt The Q motif is unique to and characteristic of the DEAD box family of RNA helicases and controls ATP binding and hydrolysis. PMID:38604460 spMss116 possesses nine conserved motifs characteristic of the DEAD box family of RNA helicases PMID:28520979 These results demonstrate that the ATP-dependent helicase activity of Mss116 is necessary for its function in mitochondrial translation. |
| GO:0003724 RNA helicase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 3.6.4.13 (RNA helicase) mapping. Consistent with the IBA and ISO rows for the same term. Reason: Same claim as the IBA and ISO rows, which are accepted; the EC assignment rests on intact DEAD-box helicase motifs. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt Belongs to the DEAD box helicase family. DDX18/HAS1 |
| GO:0003724 RNA helicase activity | ISO GO_REF:0000024 | ACCEPT | Summary: PomBase orthology transfer from S. cerevisiae Mss116 (P15424), whose RNA chaperone/helicase activity is well established and is required for its roles in intron splicing and COX1 translation. Reason: The transfer of the molecular activity is sound. The two orthologs share the full set of DEAD-box motifs and a similar predicted fold. Their biological roles have diverged (the budding-yeast protein does not complement the fission-yeast deletion), but that bears on the process annotations, not on the shared helicase activity. This is the core molecular function of the gene. Supporting Evidence: PMID:15618406 the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones PMID:28520979 These results demonstrate that the ATP-dependent helicase activity of Mss116 is necessary for its function in mitochondrial translation. PMID:38604460 spMss116 possesses nine conserved motifs characteristic of the DEAD box family of RNA helicases |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping. mss116 has the helicase ATP-binding domain with a P-loop ATP-binding site and the Q motif. Reason: Correct, but ATP binding is a component of the ATP-dependent RNA helicase activity rather than a separate function. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt /ligand="ATP" |
| GO:0005739 mitochondrion | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: Genome-wide YFP-tagging localisation screen in fission yeast detected the protein in mitochondria. This was confirmed by subcellular fractionation in PMID:38604460. The assay cannot resolve the sub-compartment. Reason: Direct fission-yeast evidence for the organelle, consistent with the predicted transit peptide. It is also confirmed by the low-throughput fractionation, protease-protection and carbonate-extraction experiments of PMID:38604460, which would support a gene-specific IDA row for the same term. Accepted under the root organelle policy; the matrix is covered by the matrix rows. Supporting Evidence: PMID:16823372 we determined the localization of 4,431 proteins PMID:38604460 Previous genome-wide protein location analysis in S. pombe found that spMss116 is located in mitochondria |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference seeded by S. cerevisiae MSS116 (SGD:S000002602), the ortholog. The organelle claim holds; the fission-yeast HDA row, the fractionation and protease-protection experiments of PMID:38604460 and the transit peptide confirm it. Reason: The node is the Mss116 mitochondrial clade, and mss116 is inside it with an intact transit peptide. The organelle claim is correct; the sub-compartment (matrix) is covered by separate rows. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt /note="Mitochondrion" PMID:38604460 spMss116 was protected from proteinase K digestion in the absence of Triton X-100 (Fig. 1B), demonstrating that spMss116 is localized inside the mitochondria. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping (Mitochondrion matrix, By similarity). Fission-yeast data place the protein inside mitochondria (protected from proteinase K in intact organelles) and mostly in the carbonate-soluble fraction, with a small membrane-associated pool. Together with the predicted cleavable transit peptide, these give two independent lines for an internal soluble location. Reason: The protease-protection assay alone cannot separate matrix from intermembrane space, because no intermembrane-space marker or mitoplast preparation was used. The cleavable N-terminal presequence, however, is the default matrix-targeting signal. All the processes that need mss116 (expression of mtDNA-encoded genes, mitoribosome assembly, processing of cox1 and cob1 transcripts) take place in the matrix. The authors' suggestion of a peripheral membrane association, from a small carbonate-pellet fraction, is compatible with a matrix-side, inner-membrane-associated pool. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion matrix {ECO:0000250}. PMID:38604460 spMss116 was protected from proteinase K digestion in the absence of Triton X-100 (Fig. 1B), demonstrating that spMss116 is localized inside the mitochondria. PMID:38604460 Most of spMss116 was found in the soluble fraction after treatment with Na2CO3, with a small amount in the pellet fraction (Fig. 1C). |
| GO:0005759 mitochondrial matrix | ISS GO_REF:0000024 | ACCEPT | Summary: PomBase transfer from S. cerevisiae Mss116, which associates with matrix mitoribosomes and acts on mitochondrial transcripts. Consistent with the fission-yeast protease-protection and carbonate-extraction data. Reason: Consistent with the transit peptide, the internal and largely soluble location seen in fission yeast, and the matrix-resident processes that require mss116. The is_active_in qualifier is reasonable because every process the protein is known to be needed for takes place in the matrix. Supporting Evidence: PMID:28520979 Mss116 co-sedimented with fully assembled ribosomes in extracts prepared in the presence of 0.5 mM MgCl2 and with dissociated mtLSU PMID:38604460 Most of spMss116 was found in the soluble fraction after treatment with Na2CO3, with a small amount in the pellet fraction (Fig. 1C). |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Rhea mapping of the generic ATP + H2O reaction attached to EC 3.6.4.13. DEAD-box helicases hydrolyse ATP, but only coupled to RNA remodelling. Reason: True as chemistry, but ATP hydrolysis is the energy-coupling step of the RNA helicase activity rather than a separate function; RNA helicase activity is the informative core term. Supporting Evidence: file:SCHPO/mss116/mss116-uniprot.txt The Q motif is unique to and characteristic of the DEAD box family of RNA helicases and controls ATP binding and hydrolysis. |
| GO:0090615 mitochondrial mRNA processing | ISS GO_REF:0000024 | MARK AS OVER ANNOTATED | Summary: Transferred from S. cerevisiae Mss116, which acts directly as an RNA chaperone for splicing of all group I and II mitochondrial introns. In fission yeast, deletion of mss116 does cause accumulation of intron-retaining cox1 and cob1 pre-mRNAs and loss of the spliced mRNAs. However, the same deletion abolishes global mitochondrial translation, which the intron-encoded maturases required for splicing depend on. The authors explicitly leave open whether mss116 participates in splicing. Reason: The full text was read; the uncertainty is direct versus indirect participation, which is a judgement, so UNDECIDED is not used. The fission-yeast splicing defect is real but is equally explained by an indirect route. Loss of mitochondrial translation removes the intron-encoded maturases, and splicing fails as a consequence. The paper does not separate the two, for example with a translation-competent allele or by measuring maturase levels. Orthology alone is a weak guide here, because the paper documents functional divergence. The budding-yeast protein cannot rescue the fission-yeast deletion, and its cox1 translational role is taken over in fission yeast by the Mrh5 complex. The annotation is not shown to be wrong, but neither a direct role nor a more specific group I or group II intron splicing term can be established from the available evidence. Supporting Evidence: PMID:38604460 RT-PCR with cox1 and cob1 intron primers revealed that the levels of intronretaining cox1 and cob1 pre-mRNAs were increased in Ξspmss116 cells PMID:38604460 relies on their intron-encoded proteins, whose synthesis was dependent on mitochondrial translation. PMID:38604460 It remains to be determined whether spMss116 participates in splicing of mitochondrial introns. PMID:15618406 Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs |
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Download this section (compressed HTML)Q: Should GO:0032543 mitochondrial translation be annotated from PMID:38604460? Global mitochondrial translation is lost in the deletion, independently of introns, and this is the paper's headline conclusion. It is not annotated here because the protein shows no association with mitoribosomes and the translation loss may follow from the assembly defect, so the evidence shows necessity rather than participation. For comparison, PomBase annotates the mitochondrial DEAD-box helicase mrh5, which binds the small subunit and activates cox1 translation, with molecular-function terms (mitochondrial small ribosomal subunit binding, translation activator activity); neither applies to mss116, which does not co-sediment with either subunit and whose defect is global.
Q: Should GO:0061668 mitochondrial ribosome assembly be annotated from PMID:38604460? The paper shows reduced assembled mitoribosomes and lower mtSSU and mtLSU protein levels in intron-containing and intronless backgrounds, with mt-rRNA unchanged. But the protein does not co-sediment with either subunit, and the authors only conclude it "may participate". In addition, S. pombe mtDNA encodes an mtSSU protein, so a primary translation defect could itself impair assembly. This review records necessity but declines to assert participation. Does PomBase hold this paper as phenotype (FYPO) annotations only, and was that deliberate?
Q: Is the accumulation of intron-retaining cox1 and cob1 pre-mRNAs a direct splicing defect or a consequence of losing the translation-dependent intron-encoded maturases? This decides whether GO:0090615 (or group I / group II intron splicing terms) should be kept for fission-yeast mss116.
Q: Why can budding-yeast Mss116 not replace fission-yeast mss116, and does this divergence argue against transferring budding-yeast process annotations (intron splicing, COX1-specific translation) to the fission-yeast protein at all?
Q: The small carbonate-resistant pool suggests a peripheral membrane association. Is fission-yeast mss116 associated with the matrix face of the inner membrane, where yeast mitoribosome assembly takes place?
Experiment: Sucrose-gradient sedimentation of mitochondrial extracts from wild-type and mss116-delta cells under milder lysis conditions or after in-organello crosslinking, probing several mtLSU and mtSSU markers and tagged spMss116, and analysing mt-rRNA by northern blot across fractions.
Hypothesis: spMss116 transiently associates with an early mitoribosome assembly intermediate, which accumulates or is degraded in its absence. Its failure to co-sediment under standard DDM/Mg2+ conditions reflects a labile interaction.
Experiment: Complement mss116-delta cells (intronless mtDNA) with ATP-binding-site (residues 83-90) and DEAD-box (195-198) point mutants of spMss116 and assay mitoribosome assembly and mitochondrial translation.
Hypothesis: The ATPase/helicase activity of spMss116 is required for mitoribosome assembly, consistent with direct remodelling of rRNA.
Experiment: In intron-containing cells, use a conditional (degron or promoter-shutoff) mss116 allele to follow cox1 and cob1 splicing and mitochondrial translation over time after depletion. Alternatively, test in vitro whether purified spMss116 stimulates splicing of the cob1 group II intron or the cox1 group I introns.
Hypothesis: If spMss116 acts directly on the introns, splicing fails before global translation declines and is stimulated by the purified protein in vitro. If it acts indirectly, splicing fails only after the maturases are depleted.
Experiment: CLIP or RNA immunoprecipitation of tagged spMss116 from mitochondria, followed by sequencing.
Hypothesis: spMss116 binds specific regions of the mitochondrial large- or small-subunit rRNA, identifying its assembly substrate.
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