The DEAD box helicase DDX6/Me31B functions in translational repression and mRNA decapping. How particular RNA helicases are recruited specifically to distinct functional complexes is poorly understood. We present the crystal structure of the DDX6 C-terminal RecA-like domain bound to a highly conserved FDF sequence motif in the decapping activator EDC3. The FDF peptide adopts an alpha-helical conformation upon binding to DDX6, occupying a shallow groove opposite to the DDX6 surface involved in RNA binding and ATP hydrolysis. Mutagenesis of Me31B shows the relevance of the FDF interaction surface both for Me31B's accumulation in P bodies and for its ability to repress the expression of bound mRNAs. The translational repressor Tral contains a similar FDF motif. Together with mutational and competition studies, the structure reveals why the interactions of Me31B with EDC3 and Tral are mutually exclusive and how the respective decapping and translational repressor complexes might hook onto an mRNA substrate.

GW182 family proteins interact directly with Argonaute proteins and are required for miRNA-mediated gene silencing in animal cells. The domains of the GW182 proteins have recently been studied to determine their role in silencing. These studies revealed that the middle and C-terminal regions function as an autonomous domain with a repressive function that is independent of both the interaction with Argonaute proteins and of P-body localization. Such findings reinforce the idea that GW182 proteins are key components of miRNA repressor complexes in metazoa.

Decapping of eukaryotic messenger RNAs (mRNAs) occurs after they have undergone deadenylation, but how these processes are coordinated is poorly understood. In this study, we report that Drosophila melanogaster HPat (homologue of Pat1), a conserved decapping activator, interacts with additional decapping factors (e.g., Me31B, the LSm1–7 complex, and the decapping enzyme DCP2) and with components of the CCR4–NOT deadenylase complex. Accordingly, HPat triggers deadenylation and decapping when artificially tethered to an mRNA reporter. These activities reside, unexpectedly, in a proline-rich region. However, this region alone cannot restore decapping in cells depleted of endogenous HPat but also requires the middle (Mid) and the very C-terminal domains of HPat. We further show that the Mid and C-terminal domains mediate HPat recruitment to target mRNAs. Our results reveal an unprecedented role for the proline-rich region and the C-terminal domain of metazoan HPat in mRNA decapping and suggest that HPat is a component of the cellular mechanism that couples decapping to deadenylation in vivo.

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...