title: MUT-14 and SMUT-1 DEAD box RNA helicases have overlapping roles in germline RNAi and endogenous siRNA formation.
abstract: More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, 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. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. 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.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Phillips
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Brooke E
authors.initials: BE
authors.lastname: Montgomery
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Breen
authors.affiliation: Institute of Molecular Biology, 55128 Mainz, Germany.
authors.forename: Elke F
authors.initials: EF
authors.lastname: Roovers
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Young-Soo
authors.initials: YS
authors.lastname: Rim
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Toshiro K
authors.initials: TK
authors.lastname: Ohsumi
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Newman
authors.affiliation: Hubrecht Institute-KNAW and University Medical Centre Utrecht, 3584 CT Utrecht, the Netherlands.
authors.forename: Josien C
authors.initials: JC
authors.lastname: van Wolfswinkel
authors.affiliation: Institute of Molecular Biology, 55128 Mainz, Germany; Hubrecht Institute-KNAW and University Medical Centre Utrecht, 3584 CT Utrecht, the Netherlands.
authors.forename: Rene F
authors.initials: RF
authors.lastname: Ketting
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02114, USA. Electronic address: ruvkun@molbio.mgh.harvard.edu.
authors.forename: Gary
authors.initials: G
authors.lastname: Ruvkun
authors.affiliation: Department of Biology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: tai.montgomery@colostate.edu.
authors.forename: Taiowa A
authors.initials: TA
authors.lastname: Montgomery
mesh_terms: D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D015200:Fluoroimmunoassay; D005854:Germ Cells / Q000201:enzymology* / Q000502:physiology; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D060888:Real-Time Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D029742:Caenorhabditis elegans Proteins; D034741:RNA, Small Interfering; D053487:DEAD-box RNA Helicases; C000600926:MUT-14 protein, C elegans; C000600927:SMUT-1 protein, C elegans