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MTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading [LARP1 KD, Torin1]

ABSTRACT: Translation of TOP mRNAs encoding protein synthesis machinery is strictly regulated by an amino acid sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino acid-rich condition, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail length regulation. Consistent with this, tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. Poly(A) tail shortens under mTOR active/ amino acid-rich condition, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino acid-starved (AAS) condition. An RNA-binding protein LARP1 that specifically binds to TOP mRNAs is indispensable for the process. We also show that LARP1 interacts with non-canonical poly(A) polymerases, PAPD4, PAPD5 and PAPD7 and induces post-transcriptional polyadenylation of the target when tethered to the mRNA. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tail under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.

ORGANISM(S): Homo sapiens

PROVIDER: GSE137807 | GEO | 2022/09/19

REPOSITORIES: GEO

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mTOR- and LARP1-dependent regulation of TOP mRNA poly(A) tail and ribosome loading [polysome fractionation]

Project description:Translation of TOP mRNAs encoding protein synthesis machinery is strictly regulated by an amino acid sensing mTOR pathway. However, its regulatory mechanism remains elusive. Here, we demonstrate that TOP mRNA translation positively correlates with its poly(A) tail length under mTOR active/amino acid-rich condition, suggesting that TOP mRNAs are post-transcriptionally controlled by poly(A) tail length regulation. Consistent with this, tail length of TOP mRNAs dynamically fluctuates in response to amino acid availability. Poly(A) tail shortens under mTOR active/ amino acid-rich condition, whereas the long-tailed TOP mRNAs accumulate under mTOR inactive/amino acid-starved (AAS) condition. An RNA-binding protein LARP1 that specifically binds to TOP mRNAs is indispensable for the process. We also show that LARP1 interacts with non-canonical poly(A) polymerases, PAPD4, PAPD5 and PAPD7 and induces post-transcriptional polyadenylation of the target when tethered to the mRNA. Our findings illustrate that LARP1 contributes to the selective accumulation of TOP mRNAs with long poly(A) tail under AAS, resulting in accelerated ribosomal loading onto TOP mRNAs for the resumption of translation after AAS.

2022-09-22 | GSE145744 | GEO

The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction

Project description:The mammalian target of rapamycin (mTOR) is a pivotal kinase responsible for transducing cellular energy signals to regulate a host of metabolic processes including protein synthesis, which in turn regulate cell growth and proliferation. All aspects of mRNA life cycle are controlled by protein/RNA interactions and although several effectors of mTOR signalling have been identified to date, how mTOR re-sculptures the mRNA interactome is unknown. Here we characterise mTOR regulated RNA-binding proteins, identifying LARP1, whose binding to RNA increases upon mTOR inhibition. We identified over 3800 LARP1 bound mRNAs, which can be broken down into two groups, those constantly bound by LARP1, or mRNAs that increase their interaction following mTOR inhibition. LARP1 has been implicated in the control of TOP mRNA translation and importantly we observe a large number of TOP mRNAs increasing association with LARP1 upon mTOR inhibition. Regarding the regulation of LARP1, we show that LARP1 and PABP show coordinated differential mRNA binding after mTOR inhibition. Importantly we find that LARP1-PABP interaction is important for LARP1 mRNA binding and mutations in the DM15 domain of LARP1 do not perturb its RNA interaction. Lastly we show that mRNAs bound by LARP1 and PABP are translationally repressed, including mRNAs encoding proteins critical for cell growth and survival.

2020-12-11 | GSE142539 | GEO

LARP1 senses free ribosomes to coordinate supply and demand of ribosomal proteins

Project description:Terminal oligopyrimidine motif-containing (TOP) mRNAs encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOP mRNAs. However, a mechanistic understanding of how LARP1 and TOP mRNAs interact with ribosomes to coordinate TOP mRNA outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating 80S ribosomes. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel and mutations at the LARP1-ribosome interface block formation of the 40S/80S-LARP1-TOP complexes. Free cytosolic ribosomes induce sequestration of TOP mRNAs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to the availability of free ribosomes.

2023-10-28 | GSE246077 | GEO

Post-transcriptional regulation of Ribosome Biogenesis in Larp1-deficient cells

Project description:Ribosome biogenesis is a critical aspect of cell differentiation. Ribosome synthesis has been previously reported to be regulated at the transcriptional and post-transcriptional levels. Poly(A) tail-length processing is a hallmark of post-transcriptional regulation associated with different steps of transcript metabolism. Here we monitor the contribution of the RNA-binding protein Larp1 in shaping the poly(A) tail profile of undifferentiated P19 cells. We found that Larp1 prevents the widespread shortening of poly(A) tails below 30 nucleotides and confers additional protection to transcripts containing a 5' terminal oligopyrimidine (TOP) motif, such as those encoding for ribosomal proteins.

2023-08-04 | GSE231976 | GEO

mTORC1 controls a dynamic program of TOP mRNA translation via LARP1

Project description:LARP1 has been proposed to control the translation of TOP mRNAs downstrteam of mTORC1. Here we used ribosome profiling to analyze transcriptome-wide changes in translation following mTOR inhibition in wild-type HEK-293T cells and cells where LARP1 (sgLARP1) or LARP1 and its homologue LARP1B (sgLARP1/1B) have been deleted using CRISPR/Cas9.

2020-02-12 | GSE132703 | GEO

GCN2 suppresses TOP mRNA translation via LARP1

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

LARP1 mRNP scaffolds mTORC1 to stimulate translation initiation of an essential class of mRNAs

Project description:The RNA biding protein, LARP1, has been proposed to function downstream of mTORC1 to positively regulate the translation of 5M-bM-^@M-^YTOP mRNAs such as ribosome protein (RP) mRNAs. However, its regulatory roles in mTORC1-mediated translation remain unclear. PAR-CLIP of LARP1 revealed its direct and dynamic interactions with RP mRNAs through pyrimidine-enriched sequences in the 5M-bM-^@M-^YUTR of RP mRNAs when mTOR activity is inhibited. Importantly, this LARP1 is a direct substrate of mTORC1 and S6K1/Akt, and phosphorylated LARP1 scaffolds mTORC1 on translation-competent mRNAs to facilitate 4EBP1 and S6K1 phosphorylation. Ablation of LARP1 causes multiple defects in the processes of translation including abnormal eIF4G1 interaction with RP mRNAs and inefficient RP mRNA elongation thereby reducing ribosome biogenesis and cell proliferation. These observations illustrate that LARP1 functions both an effector and a regulator for local mTORC1 activity, and acts as a molecular switch for ribosome biogenesis by sensing growth factor/nutrient signaling. LARP1-bound RNA regions were sequenced from HEK293T cells under growing or mTOR-inactive conditions. In parallel, mRNA abundance was quantified, in biological duplicate, from HEK293T cells under the same conditions.

2014-07-31 | E-GEOD-59599 | biostudies-arrayexpress

LARP1 mRNP scaffolds mTORC1 to stimulate translation initiation of an essential class of mRNAs

Project description:The RNA biding protein, LARP1, has been proposed to function downstream of mTORC1 to positively regulate the translation of 5’TOP mRNAs such as ribosome protein (RP) mRNAs. However, its regulatory roles in mTORC1-mediated translation remain unclear. PAR-CLIP of LARP1 revealed its direct and dynamic interactions with RP mRNAs through pyrimidine-enriched sequences in the 5’UTR of RP mRNAs when mTOR activity is inhibited. Importantly, this LARP1 is a direct substrate of mTORC1 and S6K1/Akt, and phosphorylated LARP1 scaffolds mTORC1 on translation-competent mRNAs to facilitate 4EBP1 and S6K1 phosphorylation. Ablation of LARP1 causes multiple defects in the processes of translation including abnormal eIF4G1 interaction with RP mRNAs and inefficient RP mRNA elongation thereby reducing ribosome biogenesis and cell proliferation. These observations illustrate that LARP1 functions both an effector and a regulator for local mTORC1 activity, and acts as a molecular switch for ribosome biogenesis by sensing growth factor/nutrient signaling.

2014-07-31 | GSE59599 | GEO

The 40S-LARP1 complex reprograms the cellular translatome upon mTOR inhibition to preserve the protein synthetic capacity

Project description:Ribosomes execute the transcriptional program in every cell. Critical to sustain nearly all cellular activities, ribosome biogenesis requires the translation of ~200 factors of which 80 are ribosomal proteins (RPs). As ribosome synthesis depends on RP mRNAs translation, a priority within the translatome architecture should exist to ensure the preservation of ribosome biogenesis capacity, particularly under adverse growth conditions. Here we show that under critical metabolic constraints characterized by mTOR inhibition, LARP1 complexed with the 40S subunit protects from ribophagy the mRNAs regulon for ribosome biogenesis and protein synthesis, acutely preparing the translatome to promptly resume ribosomes production after growth conditions return permissive. Characterizing the LARP1-protected translatome revealed a set of 5’TOP transcript isoforms other than RPs involved in energy production and in mitochondrial function, among other processes, indicating that the mTOR-LARP1-5’TOP axis acts at the translational level as a primary guardian of the cellular anabolic capacity

2021-10-30 | GSE186779 | GEO

mRNA-Seq of neuronal compartments in shRNA-mediated knockdown of Larp1 in mouse primary cortical neurons

Project description:To study the effect of Larp1 on the abundance and subcellular localization of 5'TOP containing mRNAs, Larp1 was depleted from mouse primary cortical neurons using shRNAs. RNA from subcellular compartments (neurite and soma cytoplasm) was isolated and sequenced in parallel with scrambled control shRNA expressing samples.

2023-07-12 | E-MTAB-12886 | biostudies-arrayexpress

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