Structure of the LSm657 complex: an assembly intermediate of the LSm1-7 and LSm2-8 rings

The nuclear LSm2-8 (like Sm) complex and the cytoplasmic LSm1-7 complex play a central role in mRNA splicing and degradation, respectively. The LSm proteins are related to the spliceosomal Sm proteins that form a heteroheptameric ring around small nuclear RNA. The assembly process of the heptameric Sm complex is well established and involves several smaller Sm assembly intermediates. The assembly of the LSm complex, however, is less well studied. Here, we solved the 2.5 Å-resolution structure of the LSm assembly intermediate that contains LSm5, LSm6, and LSm7. The three monomers display the canonical Sm fold and arrange into a hexameric LSm657-657 ring. We show that the order of the LSm proteins within the ring is consistent with the order of the related SmE, SmF, and SmG proteins in the heptameric Sm ring. Nonetheless, differences in RNA binding pockets prevent the prediction of the nucleotide binding preferences of the LSm complexes. Using high-resolution NMR spectroscopy, we confirm that LSm5, LSm6, and LSm7 also assemble into a  60-kDa hexameric ring in solution. With a combination of pull-down and NMR experiments, we show that the LSm657 complex can incorporate LSm23 in order to assemble further towards native LSm rings. Interestingly, we find that the NMR spectra of the LSm57, LSm657-657, and LSm23-657 complexes differ significantly, suggesting that the angles between the LSm building blocks change depending on the ring size of the complex. In summary, our results identify LSm657 as a plastic and functional building block on the assembly route towards the LSm1-7 and LSm2-8 complexes.     

The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies

A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182. GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs). The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit. In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182. Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1.     

Xenopus LSm proteins bind U8 snoRNA via an internal evolutionarily conserved octamer sequence

U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.     

Reconstitution of two recombinant LSm protein complexes reveals aspects of their architecture, assembly, and function

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1-7 and LSm2-8 complexes from their constituent components in vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1-7 and LSm2-8. Isolated sub-complexes form ring-like higher order structures. LSm1-7 is assembled and stable in the absence of RNA. LSm1-7 forms ring-like structures very similar to LSm2-8 at the EM level. Our in vitro reconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and an in vivo cellular transport assay.     

A Highlights from MBoC Selection: Nuclear LSm8 affects number of cytoplasmic processing bodies via controlling cellular distribution of Like-Sm proteins

Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2–7 proteins. We propose a model in which redistribution of LSm2–7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.     

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.     

The human Pat1b protein: a novel mRNA deadenylation factor identified by a new immunoprecipitation technique

The complex of the yeast Lsm1p-7p proteins with Pat1p is an important mRNA decay factor that is involved in translational shutdown of deadenylated mRNAs and thus prepares these mRNAs for degradation. While the Lsm proteins are highly conserved, there is no unique mammalian homolog of Pat1p. To identify proteins that interact with human LSm1, we developed a novel immunoprecipitation technique that yields virtually pure immunocomplexes. Mass-spec analysis therefore identifies mostly true positives, avoiding tedious functional screening. The method unambiguously identified the Pat1p homolog in HeLa cells, Pat1b. When targeted to a reporter mRNA, Pat1b represses gene expression by inducing deadenylation of the mRNAs. This demonstrates that Pat1b, unlike yPat1p, acts as an mRNA-specific deadenylation factor, highlighting the emerging importance of deadenylation in the mRNA regulation of higher eukaryotes.     

The C‐terminal α–α superhelix of Pat is required for mRNA decapping in metazoa

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N‐terminal sequence, a proline‐rich region, a Mid domain and a C‐terminal domain (Pat‐C). We show that Pat‐C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P‐rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P‐rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P‐rich region. Structural analysis revealed that Pat‐C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat‐C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.     

LSm1-7 complexes bind to specific sites in viral RNA genomes and regulate their translation and replication

LSm1-7 complexes promote cellular mRNA degradation, in addition to translation and replication of positive-strand RNA viruses such as the Brome mosaic virus (BMV). Yet, how LSm1-7 complexes act on their targets remains elusive. Here, we report that reconstituted recombinant LSm1-7 complexes directly bind to two distinct RNA-target sequences in the BMV genome, a tRNA-like structure at the 3′-untranslated region and two internal A-rich single-stranded regions. Importantly, in vivo analysis shows that these sequences regulate the translation and replication of the BMV genome. Furthermore, both RNA-target sequences resemble those found for Hfq, the LSm counterpart in bacteria, suggesting conservation through evolution. Our results provide the first evidence that LSm1-7 complexes interact directly with viral RNA genomes and open new perspectives in the understanding of LSm1-7 functions.     

Analysis of P-body assembly in Saccharomyces cerevisiae

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.     

The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.     

3'-cyclic phosphorylation of U6 snRNA leads to recruitment of recycling factor p110 through LSm proteins

Pre-mRNA splicing proceeds through assembly of the spliceosome complex, catalysis, and recycling. During each cycle the U4/U6.U5 tri-snRNP is disrupted and U4/U6 snRNA base-pairing unwound, releasing separate post-spliceosomal U4, U5, and U6 snRNPs, which have to be recycled to the splicing-competent tri-snRNP. Previous work implicated p110--the human ortholog of the yeast Prp24 protein--and the LSm2-8 proteins of the U6 snRNP in U4/U6 recycling. Here we show in vitro that these proteins bind synergistically to U6 snRNA: Both purified and recombinant LSm2-8 proteins are able to recruit p110 protein to U6 snRNA via interaction with the highly conserved C-terminal region of p110. Furthermore, the presence of a 2',3'-cyclic phosphate enhances the affinity of U6 snRNA for the LSm2-8 proteins and inversely reduces La protein binding, suggesting a direct role of the 3'-terminal phosphorylation in RNP remodeling during U6 biogenesis.     

Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development

mRNA turnover in eukaryotes involves the removal of m7GDP from the 5' end. This decapping reaction is mediated by a protein complex well characterized in yeast and human but not in plants. The function of the decapping complex in the development of multicellular organisms is also poorly understood. Here, we show that Arabidopsis thaliana DCP2 can generate from capped mRNAs, m7GDP, and 5'-phosphorylated mRNAs in vitro and that this decapping activity requires an active Nudix domain. DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS), an Arabidopsis homolog of human Hedls/Ge-1. Moreover, the interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. Consistent with their role in mRNA decay, DCP1, DCP2, and VCS colocalize in cytoplasmic foci, which are putative Arabidopsis processing bodies. Compared with the wild type, null mutants of DCP1, DCP2, and VCS accumulate capped mRNAs with a reduced degradation rate. These mutants also share a similar lethal phenotype at the seedling cotyledon stage, with disorganized veins, swollen root hairs, and altered epidermal cell morphology. We conclude that mRNA turnover mediated by the decapping complex is required for postembryonic development in Arabidopsis.     

ARE-mRNA degradation requires the 5'-3' decay pathway

As an important mode of suppressing gene expression, messenger RNAs containing an AU-rich element (ARE) in the 3' untranslated region are rapidly degraded in the cytoplasm. ARE-mediated mRNA decay (AMD) is initiated by deadenylation, and in vitro studies have indicated that subsequent degradation occurs in the 3'-5' direction through a complex of exonucleases termed the exosome. An alternative pathway of mRNA degradation occurs at processing bodies, cytoplasmic foci that contain decapping enzymes, the 5'-3' exonuclease Xrn1 and the Lsm1-7 heptamer. To determine which of the two pathways is important for AMD in live cells, we targeted components of both pathways using short interfering RNA in human HT1080 cells. We show that Xrn1 and Lsm1 are essential for AMD. On the other side, out of three exosome components tested, only knockdown of PmScl-75 caused a strong inhibition of AMD. Our results show that mammalian cells, similar to yeast, require the 5'-3' Xrn1 pathway to degrade ARE-mRNAs.     

Drosophila decapping protein 1, dDcp1, is a component of the oskar mRNP complex and directs its posterior localization in the oocyte

In Drosophila, posterior deposition of oskar (osk) mRNA in oocytes is critical for both pole cell and abdomen formation. Exon junction complex components, translational regulation factors, and other proteins form an RNP complex that is essential for directing osk mRNA to the posterior of the oocyte. Until now, it has not been clear whether the mRNA degradation machinery is involved in regulating osk mRNA deposition. Here we show that Drosophila decapping protein 1, dDcp1, is a posterior group gene required for the transport of osk mRNA. In oocytes, dDcp1 is localized posteriorly in an osk mRNA position- and dosage-dependent manner. In nurse cells, dDcp1 colocalizes with dDcp2 and Me31B in discrete foci that may be related to processing bodies (P bodies), which are sites of active mRNA degradation. Thus, as well as being a general factor required for mRNA decay, dDcp1 is an essential component of the osk mRNP localization complex.     

Nuclear pre-mRNA decapping and 5' degradation in yeast require the Lsm2-8p complex

Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)(+) RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5' degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5' capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.     

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.     

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

We describe the isolation and molecular characterization of seven distinct proteins present in human [U4/U6.U5] tri-snRNPs. These proteins exhibit clear homology to the Sm proteins and are thus denoted LSm (like Sm) proteins. Purified LSm proteins form a heteromer that is stable even in the absence of RNA and exhibits a doughnut shape under the electron microscope, with striking similarity to the Sm core RNP structure. The purified LSm heteromer binds specifically to U6 snRNA, requiring the 3'-terminal U-tract for complex formation. The 3'-end of U6 snRNA was also co-precipitated with LSm proteins after digestion of isolated tri-snRNPs with RNaseT(1). Importantly, the LSm proteins did not bind to the U-rich Sm sites of intact U1, U2, U4 or U5 snRNAs, indicating that they can only interact with a 3'-terminal U-tract. Finally, we show that the LSm proteins facilitate the formation of U4/U6 RNA duplices in vitro, suggesting that the LSm proteins may play a role in U4/U6 snRNP formation.