The LARP1 La-Module recognizes both ends of TOP mRNAs.
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ABSTRACT:
SUBMITTER: Al-Ashtal HA
PROVIDER: S-EPMC7927982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The 5'terminal oligopyrimidine (5'TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.
| S-EPMC5419741 | biostudies-literature
Project description:Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies have implicated LARP1 in 40S- or 80S-ribosome complexes that are thought to repress and stabilize TOPs. However, a molecular understanding of how LARP1 and TOPs interact with these ribosome complexes is lacking. Here, we show that LARP1 directly binds non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the mRNA channel of the 40S subunit. Increased availability of free ribosomal subunits downstream of various stresses promote 60S joining at the same interface to form LARP1-80S complexes. Simultaneously, LARP1 engages the TOP via its previously characterized La/PAM2 and DM15 domains. Contrary to expectations, ribosome binding within these complexes is not required for LARP1-mediated TOP repression or stabilization, two canonical LARP1 functions. Together, this work provides molecular insight into how LARP1 directly binds ribosomal subunits and challenges existing models describing the function of repressed LARP1-40S/80S-TOP complexes.
| S-SCDT-10_1038-S44318-024-00294-Z | biostudies-other
Project description:A central mechanism of mTOR complex 1 (mTORC1) signaling is the coordinated translation of ribosomal protein and translation factor mRNAs mediated by the 5'-terminal oligopyrimidine motif (5'TOP). Recently, La-related protein 1 (LARP1) was proposed to be the specific regulator of 5'TOP mRNA translation downstream of mTORC1, while eIF4E-binding proteins (4EBP1/2) were suggested to have a general role in translational repression of all transcripts. Here, we use single-molecule translation site imaging of 5'TOP and canonical mRNAs to study the translation of single mRNAs in living cells. Our data reveal that 4EBP1/2 has a dominant role in repression of translation of both 5'TOP and canonical mRNAs during pharmacological inhibition of mTOR. In contrast, we find that LARP1 selectively protects 5'TOP mRNAs from degradation in a transcriptome-wide analysis of mRNA half-lives. Our results clarify the roles of 4EBP1/2 and LARP1 in regulating 5'TOP mRNAs and provide a framework to further study how these factors control cell growth during development and disease.
| S-EPMC10871529 | biostudies-literature
Project description:The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.
| S-EPMC4481205 | biostudies-literature
Project description:La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.
| S-EPMC9396407 | biostudies-literature
Project description:The ribosome is an essential unit of all living organisms that commands protein synthesis, ultimately fuelling cell growth (accumulation of cell mass) and cell proliferation (increase in cell number). The eukaryotic ribosome consists of 4 ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs). Despite its fundamental role in every living organism, our present understanding of how higher eukaryotes produce the various ribosome components is incomplete. Uncovering the mechanisms utilized by human cells to generate functional ribosomes will likely have far-reaching implications in human disease. Recent biochemical and structural studies revealed La-related protein 1 (LARP1) as a key new player in RP production. LARP1 is an RNA-binding protein that belongs to the LARP superfamily; it controls the translation and stability of the mRNAs that encode RPs and translation factors, which are characterized by a 5' terminal oligopyrimidine (5'TOP) motif and are thus known as TOP mRNAs. The activity of LARP1 is regulated by the mammalian target of rapamycin complex 1 (mTORC1): a eukaryotic protein kinase complex that integrates nutrient sensing with mRNA translation, particularly that of TOP mRNAs. In this review, we provide an overview of the role of LARP1 in the control of ribosome production in multicellular eukaryotes. This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Capping and 5' End Modifications.
| S-EPMC6214789 | biostudies-literature
Project description:To investigate the changes in gene expression upon loss of LARP1, we performed RNA-seq and differential gene expression analysis on WT and derived LARP1 KO cell lines. The canonical and 5′TOP mRNA cell lines used for single-molecule imaging were treated as biological replicates for the differential gene expression analysis.
2024-01-15 | GSE233183 | GEO
Project description:The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.
| S-EPMC7269035 | biostudies-literature
Project description:The protein domain arrangement known as the La-module, comprised of a La motif (LaM) followed by a linker and RNA recognition motif (RRM), is found in seven La-related proteins: LARP1, LARP1B, LARP3 (La protein), LARP4, LARP4B, LARP6, and LARP7 in humans. Several LARPs have been characterized for their distinct activity in a specific aspect of RNA metabolism. The La-modules vary among the LARPs in linker length and RRM subtype. The La-modules of La protein and LARP7 bind and protect nuclear RNAs with UUU-3' tails from degradation by 3' exonucleases. LARP4 is an mRNA poly(A) stabilization factor that binds poly(A) and the cytoplasmic poly(A)-binding protein PABPC1 (also known as PABP). LARP1 exhibits poly(A) length protection and mRNA stabilization similar to LARP4. Here, we show that these LARP1 activities are mediated by its La-module and dependent on a PAM2 motif that binds PABP. The isolated La-module of LARP1 is sufficient for PABP-dependent poly(A) length protection and mRNA stabilization in HEK293 cells. A point mutation in the PAM2 motif in the La-module impairs mRNA stabilization and PABP binding in vivo but does not impair oligo(A) RNA binding by the purified recombinant La-module in vitro. We characterize the unusual PAM2 sequence of LARP1 and show it may differentially affect stable and unstable mRNAs. The unique LARP1 La-module can function as an autonomous factor to confer poly(A) protection and stabilization to heterologous mRNAs.
| S-EPMC7928023 | biostudies-literature
Project description:La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine tract (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5’TOP motif, resulting in the displacement of the eIF4E complex from TOP mRNAs. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. GCN2 inhibits TOP mRNA translation via ATF4-dependent transcriptional induction of LARP1 mRNAs and GCN1-mediated recruitment of LARP1 to stalled ribosomes. We performed ATF4 ChIP-seq experiments in both WT and GCN2 KO MEFs with or without leucine deprivation.
2022-08-31 | GSE166590 | GEO