Terminal oligopyrimidine (TOP) mRNAs (encoded by the TOP genes) are identified by a sequence of 6–12 pyrimidines at the 5′ end and by a growth-associated translational regulation. All vertebrate genes for the 80 ribosomal proteins and some other genes involved, directly or indirectly, in translation, are TOP genes. Among the numerous translation factors, only eEF1A and eEF2 are known to be encoded by TOP genes, most of the others having not been analyzed. Here, we report a systematic analysis of the human genes for translation factors. Our results show that: (1) all five elongation factors are encoded by TOP genes; and (2) among the initiation and termination factors analyzed, only eIF3e, eIF3f, and eIF3h exhibit the characteristics of TOP genes. Interestingly, these three polypeptides have been recently shown to constitute a specific subgroup among eIF3 subunits. In fact, eIF3e, eIF3f, and eIF3h are the part of the functional core of eIF3 that is not conserved in Saccharomyces cerevisiae. It has been hypothesized that they are regulatory subunits, and the fact that they are encoded by TOP genes may be relevant for their function.

Signaling through the mammalian target of rapamycin, complex 1 (mTORC1), positively regulates the transcription of ribosomal RNA (rRNA) and the synthesis of ribosomal proteins, thereby promoting the complex process of ribosome biogenesis. The major rRNAs are transcribed as a single precursor, which must be processed to create the 5.8S, 18S and 28S rRNAs. We used a new non-radioactive labeling approach to study the effects of rapamycin, an inhibitor of mTORC1, on rRNA synthesis. Rapamycin not only impaired synthesis of new 18S, 28S or 5S rRNA but also induced their decay. This prompted us to examine the effects of rapamycin on rRNA processing. We show that rapamycin also interferes with the processing events that generate 18S and 28S rRNA. rRNA transcription and processing occur in regions of the nucleus known as nucleoli. We find that the mTORC1 components raptor and mTOR are both present in nucleoli, where they may regulate rRNA maturation events. While rapamycin has no effect on overall nucleolar morphology or its proteome, it does induce loss of mTOR and raptor from them. These data show that mTORC1 is located in nucleoli where it acts to regulate events involved in ribosome biogenesis including the maturation of rRNA molecules.

Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs) and small non-coding RNAs (e.g., microRNAs) that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

MTORC1 (mammalian target of rapamycin complex 1) is controlled by diverse signals (e.g. hormones, growth factors, nutrients and cellular energy status) and regulates a range of processes including anabolic metabolism, cell growth and cell division. We have studied the impact of inhibiting mTOR on protein synthesis in human cells. Partial inhibition of mTORC1 by rapamycin has only a limited impact on protein synthesis, but inhibiting mTOR kinase activity causes much greater inhibition of protein synthesis. Using a pulsed stable-isotope-labelling technique, we show that the rapamycin and mTOR (mammalian target of rapamycin) kinase inhibitors have differential effects on the synthesis of specific proteins. In particular, the synthesis of proteins encoded by mRNAs that have a 5′-terminal pyrimidine tract is strongly inhibited by mTOR kinase inhibitors. Many of these mRNAs encode ribosomal proteins. mTORC1 also promotes the synthesis of rRNA, although the mechanisms involved remain to be clarified. We found that mTORC1 also regulates the processing of the precursors of rRNA. mTORC1 thus co-ordinates several steps in ribosome biogenesis.

MTORC1 [mTOR (mammalian target of rapamycin) complex 1] regulates diverse cell functions. mTORC1 controls the phosphorylation of several proteins involved in mRNA translation and the translation of specific mRNAs, including those containing a 5′-TOP (5′-terminal oligopyrimidine). To date, most of the proteins encoded by known 5′-TOP mRNAs are proteins involved in mRNA translation, such as ribosomal proteins and elongation factors. Rapamycin inhibits some mTORC1 functions, whereas mTOR-KIs (mTOR kinase inhibitors) interfere with all of them. mTOR-KIs inhibit overall protein synthesis more strongly than rapamycin. To study the effects of rapamycin or mTOR-KIs on synthesis of specific proteins, we applied pSILAC [pulsed SILAC (stable isotope-labelling with amino acids in cell culture)]. Our results reveal, first, that mTOR-KIs and rapamycin differentially affect the synthesis of many proteins. Secondly, mTOR-KIs inhibit the synthesis of proteins encoded by 5′-TOP mRNAs much more strongly than rapamycin does, revealing that these mRNAs are controlled by rapamycin-insensitive outputs from mTOR. Thirdly, the synthesis of certain other proteins shows a similar pattern of inhibition. Some of them appear to be encoded by ‘novel’ 5′-TOP mRNAs; they include proteins which, like known 5′-TOP mRNA-encoded proteins, are involved in protein synthesis, whereas others are enzymes involved in intermediary or anabolic metabolism. These results indicate that mTOR signalling may promote diverse biosynthetic processes through the translational up-regulation of specific mRNAs. Lastly, a SILAC-based approach revealed that, although rapamycin and mTOR-KIs have little effect on general protein stability, they stabilize proteins encoded by 5′-TOP mRNAs.