MUT-14 is a germline-expressed DEAD-box RNA helicase and a component of the mutator complex. This siRNA amplification module is nucleated by the Q/N-rich protein MUT-16 and assembles in perinuclear Mutator foci next to P granules. MUT-14 is recruited to Mutator foci by MUT-16. It acts redundantly with its paralog SMUT-1 and is needed for RNAi against germline-expressed genes, for silencing triggered by antisense RNA oligomers and co-suppression, for transposon silencing, and for the RdRP-dependent production of secondary 22G endo-siRNAs from thousands of germline targets. A catalytically inactive dominant-negative MUT-14 is RNAi defective in vivo, although mutator complexes containing it can still make siRNAs in vitro. This suggests that its ATP-dependent helicase activity starts amplification at an early step, possibly by recruiting or retaining target mRNAs in Mutator foci.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule keyword mapping to nucleotide binding, from the DEAD-box ATPase fold. Reason: Correct, very general consequence of the DEAD-box ATP-binding domain. |
| GO:0003676 nucleic acid binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping to nucleic acid binding, from the DEAD/DEAH box helicase domain. Reason: Correct but general; RNA helicase activity is the informative term. |
| GO:0003724 RNA helicase activity | IBA GO_REF:0000033 | ACCEPT | Summary: DEAD-box family IBA for RNA helicase activity. Reason: MUT-14 has the full DEAD-box helicase architecture (DEAD box, helicase ATP-binding and C-terminal domains, DEAD-box signature). A catalytic missense mutant acts as a dominant-negative and is RNAi defective, which supports a requirement for its enzymatic activity. Direct unwinding has not been assayed, but the inference is sound. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002774595 · PTN002774595 SUPPORTS TRANSFER Deep DEAD-box node. MUT-14 keeps the catalytic motifs and depends on them in vivo. Supporting Evidence: PMID:24684932 A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo |
| GO:0003724 RNA helicase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 3.6.4.13-based IEA for RNA helicase activity. Reason: Same conclusion as the IBA. |
| GO:0003729 mRNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: DEAD-box family IBA for mRNA binding. Reason: Plausible for a DEAD-box helicase proposed to recruit or retain target mRNAs in Mutator foci, but direct mRNA binding by MUT-14 has not been shown. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN002776867 · PTN002776867 SUPPORTS TRANSFER DEAD-box node with broad experimental mRNA binding. Untested for MUT-14. Supporting Evidence: PMID:24684932 The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs. |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro/UniRule mapping to ATP binding, from the DEAD-box helicase ATP-binding domain. Reason: Correct; DEAD-box helicases are ATP-dependent. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: DEAD-box IBA for nucleus, from DDX3/Vasa-like members that shuttle into the nucleus. Reason: Every localization study of MUT-14 places it in perinuclear cytoplasmic Mutator foci at the periphery of germline nuclei, not inside the nucleus. The nuclear assertion comes from family members with different localization. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN002774649 · PTN002774649 SUPPORTS SOURCE BUT NOT TARGET Node seeded by DDX3/Ded1-type helicases with nuclear roles. MUT-14 is seen only in perinuclear cytoplasmic Mutator foci. Supporting Evidence: PMID:22713602 Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. |
| GO:0007276 gamete generation | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the Vasa/GLH/VBH-1 germline helicase node for gamete generation. Reason: Vasa-clade helicases (Vasa, DDX4, GLH-1/2, VBH-1) have direct germ-cell development roles. The characterized function of MUT-14 is narrower, namely initiating siRNA amplification in Mutator foci for RNAi and endo-siRNA production. No direct role in gametogenesis has been reported for mut-14. Any fertility defects in mutator mutants are indirect results of lost small RNA silencing. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002774652 · PTN002774652 SUPPORTS SOURCE BUT NOT TARGET Vasa/germline-helicase node (Vasa, DDX4, GLH-1/2, VBH-1). MUT-14 has specialized for mutator-complex siRNA amplification. Supporting Evidence: PMID:24684932 The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs. |
| GO:0010629 negative regulation of gene expression | IBA GO_REF:0000033 | ACCEPT | Summary: DEAD-box IBA for negative regulation of gene expression. Reason: General but correct. MUT-14 is required for small-RNA-mediated silencing of thousands of germline targets. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN002774722 · PTN002774722 SUPPORTS TRANSFER General silencing term, consistent with the mutator function of MUT-14. Supporting Evidence: PMID:24684932 More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex |
| GO:0016787 hydrolase activity | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule mapping to hydrolase activity, from the ATP-hydrolysing helicase. Reason: Correct but uninformative parent of the helicase ATPase activity. |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the Vasa/germline helicase node for cell differentiation. Reason: Same reasoning as gamete generation. There is no evidence that MUT-14 acts directly in cell differentiation; its role is in small RNA amplification. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002774652 · PTN002774652 SUPPORTS SOURCE BUT NOT TARGET Vasa/germline-helicase node. The differentiation role is not shared by the mutator helicase MUT-14. |
| GO:0035194 regulatory ncRNA-mediated post-transcriptional gene silencing | IGI PMID:24684932 MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping ro... | ACCEPT | Summary: mut-14 smut-1 double mutants are defective in RNAi and lose endogenous siRNAs. MUT-14 acts redundantly with SMUT-1 in the mutator pathway. Reason: Core process. The mutator complex amplifies secondary siRNAs that guide WAGO-mediated post-transcriptional silencing. Supporting Evidence: PMID:24684932 Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. PMID:11809977 antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-14. |
| GO:0043186 P granule | IBA GO_REF:0000033 | MODIFY | Summary: IBA for P granule, from Vasa/GLH/LAF-1/VBH-1 members. Reason: MUT-14 is in Mutator foci, which sit next to P granules but are separate from them and do not depend on core P-granule components. Mutator focus (GO:1990633, a child of cytoplasmic ribonucleoprotein granule) is the more accurate location. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN002774723 · PTN002774723 SUPPORTS SOURCE BUT NOT TARGET Germline helicase node seeded by Vasa, LAF-1 and VBH-1 P-granule localization. MUT-14 is in the adjacent Mutator foci. Proposed replacements: mutator focus Supporting Evidence: PMID:22713602 The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. |
| GO:0043186 P granule | IEA GO_REF:0000117 | MODIFY | Summary: ARBA rule mapping to P granule. Reason: As for the IBA, MUT-14 is in Mutator foci, next to but separate from P granules. Proposed replacements: mutator focus Supporting Evidence: PMID:22713602 The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. |
| GO:1990633 mutator focus | IDA PMID:22713602 MUT-16 promotes formation of perinuclear mutator foci requir... | ACCEPT | Summary: MUT-14 is one of the six mutator proteins that localize to perinuclear Mutator foci. Reason: Core location, directly observed. Supporting Evidence: PMID:22713602 Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. |
| GO:1990633 mutator focus | IDA PMID:24684932 MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping ro... | ACCEPT | Summary: MUT-14 and SMUT-1 act in germ-cell Mutator foci. Reason: Core location, consistent with MUT-16-dependent recruitment. Supporting Evidence: PMID:24684932 The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. PMID:30036386 In brief, MUT-16 is required for localization of MUT-2, MUT-7, RDE-2, MUT-14, and MUT-15, all of which localize independently of one another except for MUT-7, which requires RDE-2 for localization (Fig 1C). |
| GO:0030422 siRNA processing | IGI PMID:24684932 MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping ro... | NEW | Summary: mut-14 smut-1 double mutants cause widespread loss of endogenous siRNAs, comparable to mut-16. MUT-14 is proposed to start siRNA amplification in Mutator foci. Reason: MUT-14 is a catalytic subunit of the mutator amplification complex, and its helicase activity is needed in vivo, so it contributes enzymatic work to secondary siRNA biogenesis rather than being only required for it. For the comparator check, the other mutator-complex members in the same role are MUT-16 (IMP), RDE-2 (IDA), NYN-1/NYN-2 (IGI) and RDE-8 (IMP), and all of them carry GO:0030422 in WormBase/GOA. Supporting Evidence: PMID:24684932 Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. PMID:24684932 the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation |
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Download this section (compressed HTML)Q: Which RNA substrates does MUT-14 unwind or remodel in Mutator foci, and does its ATPase cycle control how target mRNAs are captured or released for RdRP-templated 22G-RNA synthesis?
Experiment: Purify MUT-14, wild type and the catalytic mutant, and measure its ATPase and unwinding activity on target mRNA/siRNA duplexes. Use CLIP in germlines to map which mRNAs MUT-14 binds in vivo, with and without SMUT-1.
mut-14 encodes a DEAD box RNA helicase; MUT-14 activity is required redundantly with its paralog SMUT-1, for germline RNAi and endogenous siRNA formation; mut-14 mutant animals exhibit germline transposon activation, a deficient response to RNAi, resistance to co-suppression, and transgene desilencing.
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