Lsm2 and Lsm3 bridge the interaction of the Lsm1-7 complex with Pat1 for decapping activation.
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ABSTRACT:
SUBMITTER: Wu D
PROVIDER: S-EPMC3915908 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Project description:A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5'-to-3' exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7-Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7-Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7-Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7-Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7-Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7-Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.
| S-EPMC4138329 | biostudies-literature
Project description:The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.
| S-EPMC4509936 | biostudies-literature
Project description:RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under osmotic stress in pat1 and lsm1 mutants, which correlated with an abnormally high association of both non-stress and stress-induced mRNAs to translationally active polysomes. Additionally, for stress-induced proteins normally triggered only by moderate or high osmostress, in the mutants the protein levels rose high already at weak hyperosmosis. Analysis of ribosome passage on mRNAs through co-translational decay from the 5' end (5P-Seq) showed increased ribosome accumulation in lsm1 and pat1 mutants upstream of the start codon. This effect was particularly strong for mRNAs induced under osmostress. Thus, our results indicate that, in addition to its role in degradation, the Lsm1-7/Pat1 complex acts as a selective translational repressor, having stronger effect over the translation initiation of heavily expressed mRNAs. Binding of the Lsm1-7/Pat1p complex to osmostress-induced mRNAs mitigates their translation, suppressing it in conditions of weak or no stress, and avoiding a hyperresponse when triggered.
| S-EPMC6085073 | biostudies-literature
Project description:Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.
| S-EPMC5554784 | biostudies-literature
Project description:Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.
| S-EPMC7491322 | biostudies-literature
Project description:In Saccharomyces cerevisiae, mRNA transcripts with premature termination codons are targeted for deadenylation independent decapping and 5' to 3' decay in a quality control pathway termed nonsense-mediated decay (NMD). Critical factors in NMD include Upf1, Upf2, and Upf3, as well as the decapping enzyme, Dcp2/Dcp1. Loss of Upf2 or Upf3 leads to the accumulation of not only Upf1 and Dcp2 in P-bodies, but also of the decapping-activators Pat1, Dhh1, and Lsm1. An interaction between Upf1 and Dcp2 has been identified, which might recruit Dcp2 to the NMD decapping complex. To determine the nature and significance of the Dcp2-Upf1 interaction, we utilized the yeast two-hybrid assay to assess Upf1 interactions with various mRNA decapping factors. We find that although Dcp2 can interact with Upf1, this interaction is indirect and is largely dependent on the Edc3 protein, which interacts with the N-terminal domain of Upf1 at an overlapping, but not identical, site as Upf2. We also found that Pat1 has an independent two-hybrid interaction with the N-terminus of Upf1. Assessment of both reporter and endogenous NMD transcripts suggest that the decapping stimulators, including Edc3 and Pat1, as well as Edc1 and Edc2, are not essential for NMD under normal conditions. This work defines a larger decapping complex involved in NMD, but indicates that components of that complex are not required for general NMD and might either regulate a subset of NMD transcripts or be essential for proper NMD under different environmental conditions.
| S-EPMC3204985 | biostudies-literature
Project description:Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins.
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Project description:In this study, we aim to understand how the fates of stress-activated mRNAs are determined under stress conditions by isolating individual osmostress-activated mRNA species, quantitating the proteins associated in vivo with each of them, and analyzing how deletion of these proteins impact on the expression of stress-activated genes. By comparison with the proteome associated with individual not stress-related mRNA species, we show specific proteins to be preferentially binding to osmostress-activated mRNAs, notably members of the cytoplasmic Pat1 / Lsm1 – 7 complex. We evaluated the impact of this complex on the stress response by analyzing expression of osmostress-activated genes, production of osmo-protein and, mapping ribosome transit at single nucleotide resolution using 5’-phosphate sequencing of mRNAs in lsm1 and pat1 mutants. We conclude that our biochemical co-purification approach has successfully identified RNA-binding proteins with a particular role in regulating post-transcriptional expression of stress-activated mRNAs.
2018-07-18 | GSE107250 | GEO
Project description:Despite many studies on host or viral gene expression, how the cellular proteome responds to internal or external cues during the infection process remains unclear. In this study, we used a Hepatitis B Virus (HBV) replication model and performed proteomic analyses to understand how HBV evades innate immunity as a function of cell cycle progression. Specifically, we performed proteomic analyses of HBV-replicating cells in G1/S and G2/M phases, as a function of IFN-α treatment. We identified that the conserved LSm (Like-Sm1-8) proteins were differentially regulated in HBV replicating cells treated with IFN-α. Specifically, in G2/M phase, IFN-α increased protein level of LSm1, the unique subunit of cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-α decreased LSm8, the unique subunit of nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA, suggesting the cytoplasmic LSm1-7 complex is antiviral, whereas the nuclear LSm2-8 complex is pro-viral. In HBV replication and infection models, siRNA-mediated knockdown of LSm1 increased all viral RNAs. Conversely, LSm8 knockdown reduced viral RNA levels, dependent on N6-adenosine methylation (m6A) of the epsilon stem-loop at the 5' end of pre-Core/pregenomic (preC/pg) RNA. Methylated RNA immunoprecipitation (MeRIP) assays demonstrated reduced viral RNA methylation by LSm8 knockdown, dependent on the 5' m6A modification, suggesting the LSm2-8 complex has a role in mediating this modification. Interestingly, splicing inhibitor Cp028 acting upstream of the LSm2-8 complex suppressed viral RNA levels without reducing the 5' m6A modification. This observation suggests Cp028 has novel antiviral effects, likely potentiating IFN-α-mediated suppression of HBV biosynthesis.
| S-EPMC9396650 | biostudies-literature
Project description:We report the role of LSM1-7 complex in the Arabidopsis tolerance to abiotic stresses. LSM1-7 controls gene expression reprogramming at the post-transcriptional level by promoting the decapping of mRNA. This function is selectively achieve over selected stress-induced transcripts depending on stress nature.
2016-01-21 | GSE70491 | GEO