Abstract
The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5′ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.
Highlights
The mammalian target of rapamycin is the catalytic component of two multi-protein complexes known as mTORC1 and mTORC2 with differing activities dependent upon its interacting partners [1,2,3,4,5,6,7,8]. mTORC1 and mTORC2 are activated by upstream growth signals and control the balance between catabolic and anabolic processes through the phosphorylation of distinct substrates. mTORC1, in particular, plays a major role in the regulation of protein synthesis. mTORC1 mediated regulation of protein synthesis is achieved via several mechanisms [12,13]
The mammalian target of rapamycin is the catalytic component of two multi-protein complexes known as mTORC1 and mTORC2 with differing activities dependent upon its interacting partners [1,2,3,4,5,6,7,8]. mTORC1 and mTORC2 are activated by upstream growth signals and control the balance between catabolic and anabolic processes through the phosphorylation of distinct substrates (reviewed in [9,10]). mTORC1, in particular, plays a major role in the regulation of protein synthesis (reviewed in [11,12,13,14]). mTORC1 mediated regulation of protein synthesis is achieved via several mechanisms [12,13]
Northern blot analysis of sucrose density gradients demonstrated that TOP-containing mRNAs including eukaryotic elongation factor 2 (eEF2), RPS16 and RPL24 all shifted to the sub-polysomes upon mTOR inhibition, as expected [70], with less of a change in the control -actin transcript, a non-TOP mRNA (Figure 1A)
Summary
The mammalian target of rapamycin (mTOR) is the catalytic component of two multi-protein complexes known as mTORC1 and mTORC2 with differing activities dependent upon its interacting partners [1,2,3,4,5,6,7,8]. mTORC1 and mTORC2 are activated by upstream growth signals and control the balance between catabolic and anabolic processes through the phosphorylation of distinct substrates (reviewed in [9,10]). mTORC1, in particular, plays a major role in the regulation of protein synthesis (reviewed in [11,12,13,14]). mTORC1 mediated regulation of protein synthesis is achieved via several mechanisms [12,13]. MTORC1 mediated regulation of protein synthesis is achieved via several mechanisms [12,13] This includes the control of cap-dependent translation through the phosphorylation of the 4E binding proteins (4E-BP1–3 [15,16]) to regulate eIF4F complex formation, the activation of ribosomal protein S6 kinases (S6Ks) [17], which phosphorylate several substrates involved in translation elongation including the ribosomal protein S6 [18,19,20,21,22], and the eukaryotic elongation factor 2 kinase (eEF2K), which in turn modulates elongation rates through the phosphorylation of the eukaryotic elongation factor 2 (eEF2) [23].
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