Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) are present. These antibodies cause loss of AChR numbers and function, and lead to failure of neuromuscular transmission with muscle weakness. The pathogenic mechanisms acting in the 20% of patients with generalized MG who are seronegative for AChR-antibodies (AChR-Ab) have not been elucidated, but there is evidence that they also have an antibody-mediated disorder, with the antibodies directed towards another, previously unidentified muscle-surface-membrane target. Here we show that 70% of AChR-Ab-seronegative MG patients, but not AChR-Ab-seropositive MG patients, have serum auto-antibodies against the muscle-specific receptor tyrosine kinase, MuSK. MuSK mediates the agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction. The MuSK antibodies were specific for the extracellular domains of MuSK expressed in transfected COS7 cells and strongly inhibited MuSK function in cultured myotubes. Our results indicate the involvement of MuSK antibodies in the pathogenesis of AChR-Ab-seronegative MG, thus defining two immunologically distinct forms of the disease. Measurement of MuSK antibodies will substantially aid diagnosis and clinical management.

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182-AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine-tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.

Precise epithelial tube diameters rely on coordinated cell shape changes and apical membrane enlargement during tube growth. Uniform tube expansion in the developing Drosophila trachea requires the assembly of a transient intraluminal chitin matrix, where chitin forms a broad cable that expands in accordance with lumen diameter growth. Like the chitinous procuticle, the tracheal luminal chitin cable displays a filamentous structure that presumably is important for matrix function. Here, we show that knickkopf (knk) and retroactive (rtv) are two new tube expansion mutants that fail to form filamentous chitin structures, both in the tracheal and cuticular chitin matrices. Mutations in knk and rtv are known to disrupt the embryonic cuticle, and our combined genetic analysis and chemical chitin inhibition experiments support the argument that Knk and Rtv specifically assist in chitin function. We show that Knk is an apical GPI-linked protein that acts at the plasma membrane. Subcellular mislocalization of Knk in previously identified tube expansion mutants that disrupt septate junction (SJ) proteins, further suggest that SJs promote chitinous matrix organization and uniform tube expansion by supporting polarized epithelial protein localization. We propose a model in which Knk and the predicted chitin-binding protein Rtv form membrane complexes essential for epithelial tubulogenesis and cuticle formation through their specific role in directing chitin filament assembly.

Trailer Hitch (Tral or LSm15) and enhancer of decapping-3 (EDC3 or LSm16) are conserved eukaryotic members of the (L)Sm (Sm and Like-Sm) protein family. They have a similar domain organization, characterized by an N-terminal LSm domain and a central FDF motif; however, in Tral, the FDF motif is flanked by regions rich in charged residues, whereas in EDC3 the FDF motif is followed by a YjeF_N domain. We show that in Drosophila cells, Tral and EDC3 specifically interact with the decapping activator DCP1 and the DEAD-box helicase Me31B. Nevertheless, only Tral associates with the translational repressor CUP, whereas EDC3 associates with the decapping enzyme DCP2. Like EDC3, Tral interacts with DCP1 and localizes to mRNA processing bodies (P bodies) via the LSm domain. This domain remains monomeric in solution and adopts a divergent Sm fold that lacks the characteristic N-terminal ␣-helix, as determined by nuclear magnetic resonance analyses. Mutational analysis revealed that the structural integrity of the LSm domain is required for Tral both to interact with DCP1 and CUP and to localize to P-bodies. Furthermore, both Tral and EDC3 interact with the C-terminal RecA-like domain of Me31B through their FDF motifs. Together with previous studies, our results show that Tral and EDC3 are structurally related and use a similar mode to associate with common partners in distinct protein complexes.Proteins of the (L)Sm (Sm and Like-Sm) family are found in all domains of life and play critical roles in RNA metabolism (reviewed in references 26 and 54). These proteins have the Sm fold, which comprises an N-terminal ␣-helix stacked on top of a five-stranded ␤-barrel-like structure (10,25,38,44,48,49). Sm domains often oligomerize to form hexameric or heptameric rings that stably or transiently associate with singlestranded RNA. Eubacterial and archeal genomes encode between 1 and 3 LSm paralogs, which form monohexameric or monoheptameric rings, while eukaryotes encode more than 18 (L)Sm paralogs that assemble into heteroheptameric rings of different composition and function (reviewed in references 26 and 54).Although much is known about (L)Sm proteins consisting of a single Sm domain, proteins possessing an N-terminal Sm domain followed by C-terminal extensions with additional domains are less well characterized. These include LSm12 to LSm16 (1, 2). LSm12 is characterized by a C-terminal protein methyltransferase domain (1, 2). The LSm13-16 proteins share a divergent form of the Sm domain and a central FDF motif (1, 2). The FDF motifs of LSm13-15 are embedded in low-complexity regions rich in glycine and arginine (1, 2). In contrast, in LSm16 (known as enhancer of decapping-3 and referred to as EDC3 hereafter) the FDF motif is followed by a conserved C-terminal YjeF_N domain that adopts a divergent Rossman fold similar to one in the N-terminal domain of bacterial YjeF (1, 2, 32). EDC3 (LSm16) is known to enhance bulk mRNA decapping in yeast and is required for the decapping-dependent regulation of RPS28B mRNA and YRA1 pre-mRNA ...

Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal ␣-helix and has a disrupted ␤4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.Proteins of the Sm and Sm-like family [conjointly referred to as (L)Sm proteins] are found in all domains of life and play important roles in RNA processing and decay (reviewed in references 21 and 46). They share the Sm fold, which comprises an N-terminal ␣-helix stacked on top of a strongly bent, five-stranded antiparallel ␤-sheet, which forms a barrel-like structure. The fold can be divided into two segments corresponding to the highly conserved Sm1 and Sm2 motifs, where Sm1 comprises ␤-strands 1 to 3 (␤1-3) and Sm2 comprises ␤-strands 4 and 5 (␤4-5). The two motifs are joined by a nonconserved linker (L4) of variable length (18,20,34) (see Fig. 4 and 5).The (L)Sm domains often oligomerize to form hexameric or heptameric rings that stably or transiently bind single-stranded RNA. The major contacts between the subunits of the ring are mediated by antiparallel interactions between the backbones of strand ␤4 of one subunit and strand ␤5 of the adjacent subunit. RNA binding is mediated mainly by residues in loops between strands ␤2 and ␤3 and between strands ␤4 and ␤5 (loops L3 and L5, respectively), which face the lumen of the ring (7,20,26,33,40,41).The eubacterial and archaeal genomes encode from one to three (L)Sm paralogs that form homohexameric or homoheptameric rings, while eukaryotes encode more than eighteen (L)Sm paralogs that assemble into heteroheptameric rings of different composition and function (reviewed in references 1, 2, 21, and 46).Seven of the eukaryotic proteins (SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG) form a ring that stably associates with RNA polymerase II-transcribed uridine-rich small nuclear RNAs (i.e., U1, U2, U4, and U5), and functions in uridine-rich snRNP biogenesis and mRNA splicing (18,21,34,46). In addition to the S...