Similar Modes of Interaction Enable Trailer Hitch and EDC3 To Associate with DCP1 and Me31B in Distinct Protein Complexes

Abstract

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 ...

Arabidopsis Decapping 5 Is Required for mRNA Decapping, P-Body Formation, and Translational Repression during Postembryonic Development

“…The Lsm domain in Tral also mediates the interaction between Tral and DCP1. However, Tral was found not to associate with DCP2 in Drosophila, whereas Edc3 interacts with DCP2 though a linker region between the Lsm domain and the FDF motif (Tritschler et al, 2007(Tritschler et al, , 2008. However, the exact function of these Lsm domain proteins in both P-body formation and mRNA decapping is largely unknown.…”

Cytoplasmic mRNP granules at a glance

“…Key factors that promote the assembly of mRNPs into PBs include decapping factors and associated proteins that repress translation initiation, and activate decapping and 5¢ to 3¢ decay of the mRNA (see Poster) (for reviews, see Coller and Parker, 2004;Eulalio et al, 2007a;Franks and Lykke-Andersen, 2008;Simon et al, 2006). Evidence suggests that multiple alternative complexes between these factors exist that, depending on the specific complex composition and on cell conditions, either promote the repression of translation initiation only or additionally activate 5¢ to 3¢ decay of target mRNAs (Decker et al, 2007;Haas et al, 2010;Pilkington and Parker, 2008;Tritschler et al, 2009;Tritschler et al, 2008;Yoon et al, 2010). Several lines of evidence suggest that recruitment of these 5¢ repressiondecay complexes allows mRNP assembly into PBs (Anderson and Kedersha, 2009;Balagopal and Parker, 2009;Eulalio et al, 2007a;Franks and Lykke-Andersen, 2008;Kulkarni et al, 2010).…”

Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation

“…These included Me31B, the orthologs of which associate with the CCR4-NOT complex in yeast (Hata et al 1998;Maillet and Collart 2002) and in trypanosomes (Schwede et al 2008). Me31B is known to associate with either Tral or Edc3 in two mutually exclusive complexes, the former of which also contains Cup (Tritschler et al 2008). All three proteins were present in the immunoprecipitate, although a lower level of Tral and Edc3 (as judged by sequence coverage) was also present in the negative control.…”

“…All constructs were verified by sequencing. The GFP-Tral expressing plasmid (Tritschler et al 2008) was a gift from Elisa Izaurralde (Max Planck Institute for Developmental Biology). dsRNAs used for RNAi were generated as described (Bönisch et al 2007).…”