All translation elongation factors and the e, f, and h subunits of translation initiation factor 3 are encoded by 5'-terminal oligopyrimidine (TOP) mRNAs.

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.

Effect of 3'UTR length on the translational regulation of 5'-terminal oligopyrimidine mRNAs.

In Vertebrates, all genes coding for ribosomal proteins, as well as those for other proteins implicated in the production and function of translation machinery, are regulated by mitogenic and nutritional stimuli, at the translational level. A cis-regulatory element necessary for this regulation is the typical 5'UTR, common to all ribosomal protein mRNAs, which always starts at the 5' end with several pyrimidines. Having noticed that the 3'UTR of all ribosomal protein mRNAs is much shorter than most cellular mRNAs, we have now studied the possible implication of this 3'UTR feature in the translational regulation. For this purpose, we constructed a number of chimeric genes whose transcribed mRNAs contain: (1) the 5'UTR of ribosomal protein S6 mRNA or, as a control, of beta-actin mRNA; (2) the EGFP reporter coding sequence from the starting AUG to the stop codon; (3) different 3'UTRs of various lengths. These constructs have been stably transfected in human HEK293 cells, and the translation regulation of the expressed chimeric mRNAs has been analyzed for translation efficiency, in growing and in serum starved cells, by the polysome association assay. The results obtained indicate that, while the typical growth-associated translational regulation is bestowed on an mRNA by the pyrimidine sequence containing 5'UTR, the stringency of regulation depends on the short size of the 3'UTR.

RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis.

RACK1 has been shown to interact with several proteins, this suggesting that it may play a central role in cell growth regulation. Some recent articles have described RACK1 as a component of the small ribosomal subunit. To investigate the relationship between RACK1 and ribosome, we analyzed RACK1 mRNA structure and regulation. Translational regulation was studied in HeLa cells subjected to serum or amino acid deprivation and stimulation. The results show that RACK1 mRNA has a 5' terminal oligopyrimidine sequence and that its translation is dependent on the availability of serum and amino acids in exactly the same way as any other vertebrate ribosomal protein mRNA.

TOP promoter elements control the relative ratio of intron-encoded snoRNA versus spliced mRNA biosynthesis.

In vertebrates almost all snoRNAs are encoded in introns of a specific subclass of polII transcripts: the TOP genes. The majority of these RNAs originate through debranching of the spliced introns, the rest through endonucleolytic cleavage of the precursor that contains them. In both cases it has been suggested that snoRNP factors associate at early steps during transcription and control snoRNA biogenesis. Here, we analyzed the specific case of the U16 snoRNA that was shown to originate mainly through endonucleolytic cleavage. We show that TOP promoter elements determine a specific ratio of snoRNA and mRNA production. Under the control of these sequences the snoRNA is likely to originate from both splicing and cleavage of the pre-mRNA. Conversely, canonical polII promoter elements seem not to be compatible with snoRNA release through the cleavage reaction and produce a lower snoRNA/mRNA ratio. In addition, we show that the proximal part of the TOP promoter is responsible for this peculiar post-transcriptional process that controls the relative ratio between snoRNA and mRNA.

Synthesis and function of ribosomal proteins--fading models and new perspectives.

The synthesis of ribosomal proteins (RPs) has long been known to be a process strongly linked to the growth status of the cell. In vertebrates, this coordination is dependent on RP mRNA translational efficiency, which changes according to physiological circumstances. Despite many years of investigation, the trans-acting factors and the signaling pathways involved in this regulation are still elusive. At the same time, however, new techniques and classic approaches have opened up new perspectives as regards RP regulation and function. In fact, the proteasome seems to play a crucial and unpredicted role in regulating the availability of RPs for subunit assembly. In addition, the study of human ribosomal pathologies and animal models for these diseases has revealed that perturbation in the synthesis and/or function of an RP activates a p53-dependent stress response. Surprisingly, the effect of the ribosomal stress is more dramatic in specific physiological processes: hemopoiesis in humans, and pigmentation in mice. Moreover, alteration of each RP impacts differently on the development of an organism.

Translational regulation of terminal oligopyrimidine mRNAs induced by serum and amino acids involves distinct signaling events.

Various mitogenic or growth inhibitory stimuli induce a rapid change in the association of terminal oligopyrimidine (TOP) mRNAs with polysomes. It is generally believed that such translational control hinges on the mammalian target of rapamycin (mTOR)-S6 kinase pathway. Amino acid availability affects the translation of TOP mRNAs, although the signaling pathway involved in this regulation is less well characterized. To investigate both serum- and amino acid-dependent control of TOP mRNA translation and the signaling pathways involved, HeLa cells were subjected to serum and/or amino acid deprivation and stimulation. Our results indicate the following. 1). Serum and amino acid deprivation had additive effects on TOP mRNA translation. 2). The serum content of the medium specifically affected TOP mRNA translation, whereas amino acid availability affected both TOP and non-TOP mRNAs. 3). Serum signaling to TOP mRNAs involved only a rapamycin-sensitive pathway, whereas amino acid signaling depended on both rapamycin-sensitive and rapamycin-insensitive but wortmannin-sensitive events. 4). Eukaryotic initiation factor-2alpha phosphorylation increased during amino acid deprivation, but not following serum deprivation. Interestingly, rapamycin treatment suggests a novel connection between the mTOR pathway and eukaryotic initiation factor-2alpha phosphorylation in mammalian cells, which may not, however, be involved in TOP mRNA translational regulation.

Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations.

The Fragile X mental retardation gene (FMR1) contains a polymorphic trinucleotide CGG repeat in the 5' untranslated region (UTR) of the FMR1 messenger. We have characterized three lymphoblastoid cell lines derived from unrelated male carriers of a premutation that overexpress FMR1 mRNA and show reduced FMRP level compared to normal cells. The analysis of polysomes/mRNPs distribution of mRNA in the cell lines with a premutation shows that the polysomal association of FMR1 mRNA, which is high in normal cells, becomes progressively lower with increasing CGG repeat expansion. In addition, we could detect a very low level of FMR1 mRNA in a lymphoblastoid cell line from a patient with a full mutation. In this case, FMR1 mRNA is not at all associated with polysomes, in agreement with the complete absence of FMRP. The impairment of FMR1 mRNA translation in patients with the Fragile X syndrome with FMR1 premutation is the cause of the lower FMRP levels that leads to the clinical involvement.