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

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. Overall design: 5' capped polyA RNAs purified from free, monosomal, light-polysomal and heavy-polysomal fractions were analyzed by direct RNA-sequencing using the Oxford Nanopore's MinION.