A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

Transcription inhibitors stimulate translation of 5' TOP mRNAs through activation of S6 kinase and the mTOR/FRAP signalling pathway.

We have analysed the effect of transcription inhibitors on the polysomal localization of 5' terminal oligopyrimidine (TOP-) mRNAs. It is known that, in vertebrates, the translation of this group of mRNAs is regulated according to the growth status of the cell. Mitogenic stimulation of quiescent cells induces a rapid recruitment of TOP mRNAs from translationally inactive light messenger ribonucleoprotein particles to polysomes. It was found that administration of transcription inhibitors to resting cells causes a similar collective translational activation of TOP mRNAs, without affecting global translation. A number of transcription inhibitors were tested in amphibian and mammalian cultured cells. Actinomycin D (act D), cordycepin, and 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole caused a similar activation whereas alpha-amanitin or low doses of act D did not induce the translational response. Concentrations of act D sufficient to induce TOP mRNA translation also induce 40S ribosomal protein S6 kinases 1 (S6K1) activation. Moreover at these concentrations of act D increased phosphorylation of 4E-BP1 was also observed, indicating the involvement of FRAP/mTOR. Consistent with this observation, pretreatment of resting cells with rapamycin suppresses the activation of TOP mRNA translation induced by act D. These results indicate that the effect of act D on translation is mediated by the S6Ks through FRAP/mTOR.

La protein has a positive effect on the translation of TOP mRNAs in vivo.

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5'-untranslated region (5'-UTR), including a stretch of pyrimidines at the 5'-end. The 5'-terminal oligopyrimidine (5'-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5'-UTR downstream from the 5'-TOP sequence.

Chemical stimulation of synaptosomes modulates alpha -Ca2+/calmodulin-dependent protein kinase II mRNA association to polysomes.

The presence of specific mRNAs in dendrites and at synapses is well established, but a direct and reliable demonstration that they are associated with polysomes is still missing. To address this point we analyzed the polysomal association of the mRNAs for the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (alpha-CaMKII), for type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) and for the activity-regulated cytoskeleton-associated protein (Arc) in a synaptosomal preparation devoid of contaminating material from neuronal and glial perikarya. We show that a fraction of alpha-CaMKII, InsP3R1, and Arc mRNAs present in synaptosomes is indeed associated with polysomes. Moreover, we show that polysomal association of alpha-CaMKII mRNA, but not InsP3R1 and Arc mRNAs, increases with depolarization of the synaptosomal membrane. Finally, we show that the synthesis of alpha-CaMKII protein increases with stimulation. Dendritic mRNA recruitment onto polysomes in response to synaptic stimulation might represent one of the mechanisms underlying the processes of learning and memory.

Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin-hinge-hairpin-tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

Expression of the gene for mitoribosomal protein S12 is controlled in human cells at the levels of transcription, RNA splicing, and translation.

The human gene RPMS12 encodes a protein similar to bacterial ribosomal protein S12 and is proposed to represent the human mitochondrial orthologue. RPMS12 reporter gene expression in cultured human cells supports the idea that the gene product is mitochondrial and is localized to the inner membrane. Human cells contain at least four structurally distinct RPMS12 mRNAs that differ in their 5'-untranslated region (5'-UTR) as a result of alternate splicing and of 5' end heterogeneity. All of them encode the same polypeptide. The full 5'-UTR contains two types of sequence element implicated elsewhere in translational regulation as follows: a short upstream open reading frame and an oligopyrimidine tract similar to that found at the 5' end of mRNAs encoding other growth-regulated proteins, including those of cytosolic ribosomes. The fully spliced (short) mRNA is the predominant form in all cell types studied and is translationally down-regulated in cultured cells in response to serum starvation, even though it lacks both of the putative translational regulatory elements. By contrast, other splice variants containing one or both of these elements are not translationally regulated by growth status but are translated poorly in both growing and non-growing cells. Reporter analysis identified a 26-nucleotide tract of the 5'-UTR of the short mRNA that is essential for translational down-regulation in growth-inhibited cells. Such experiments also confirmed that the 5'-UTR of the longer mRNA variants contains negative regulatory elements for translation. Tissue representation of RPMS12 mRNA is highly variable, following a typical mitochondrial pattern, but the relative levels of the different splice variants are similar in different tissues. These findings indicate a complex, multilevel regulation of RPMS12 gene expression in response to signals mediating growth, tissue specialization, and probably metabolic needs.

Isolation, structural analysis and mapping of the functional gene of human ribosomal protein S26.

The nucleotide sequence of the gene of human ribosomal protein S26 has been assembled from cDNA and genomic PCR-amplified DNA fragments, and its transcription start site has been determined by primer extension. The gene is composed of four exons and three introns spanning 2027bp. Like other ribosomal protein genes of vertebrates, this gene contains a short first exon corresponding exactly to the short untranslated 5'- UTR. Its transcription start site is embedded in a polypyrimidine tract. Using PCR on DNAs from hybrid cell lines with a different set of human chromosomes, the intron-containing gene of ribosomal protein S26 was mapped to human chromosome 12.

[Cloning and structure-function analysis of the human S26 ribosomal protein gene]. / Klonirovanie i strukturno-funktsional'nyi analiz gena ribosomnogo belka S26 cheloveka.

Gene HRPS26 encoding the S26 human ribosomal protein has been sequenced. Gene HRPS26 consists of four exons and three introns. Its size is 2027 bp; the size of its mRNA is 438 nucleotides. As most of the genes of ribosomal proteins of vertebrates, the HRPS26 gene has a short first exon, which corresponds to the 5'-untranslated region of mRNA; the origin of transcription is located in the polypyrimidine tract. The functional activity of the cloned HRPS26 promoter region has been confirmed by transcription of hybrid plasmids in HeLa cells.

Translational control of terminal oligopyrimidine mRNAs requires a specific regulator.

Terminal oligopyrimidine (TOP) mRNAs are a group of messengers translationally regulated according to the growth status of the cell. Two hypotheses have been proposed for the mechanism of the regulation: (i) there is a specific translational regulator which can reversibly alter TOP-mRNA structure, (ii) a component of the general translational apparatus can specifically affect the translation of TOP-mRNAs. To verify one of the two hypotheses we induced a partial inhibition of translation initiation in Xenopus cultured cells and analyzed the effect on TOP-mRNA translation. Our results suggest that a specific regulator is necessary to explain the translational control of these of mRNAs.

Growth-dependent and growth-independent translation of messengers for heterogeneous nuclear ribonucleoproteins.

The hnRNP A1 transcript has a relatively short 5'- untranslated region (UTR) starting with a pyrimidine tract similar to that of mRNAs encoded by the TOP [terminal oligo(pyrimidine)] genes in vertebrates. Such genes code for ribosomal proteins and for other proteins directly or indirectly involved in the production and function of the translation apparatus. As expected from the role of the pyrimidine tract in the translational regulation of TOP mRNAs, the A1 mRNA is more efficiently loaded onto polysomes in growing than in resting cells. On the other hand, a less stringent regulation with respect to that of other TOP mRNAs is observed, partially due to the presence of multiple transcription start sites within the pyrimidine tract, where transcripts with shorter TOP sequences are less sensitive to regulation. Thus, from the point of view of structural features and translation behaviour the A1 mRNA can be included in the class of TOP genes, suggesting a possible role of A1 in translation. Interestingly, a TOP-like behaviour was observed for hnRNP I mRNA but not for hnRNP C1/C2 and A2/B1 mRNAs, indicating the existence of two classes of hnRNPs with different translational regulation.

Small nucleolar RNAs and nucleolar proteins in Xenopus anucleolate embryos.

We investigated the presence and localization, in the cells of anucleolate mutant embryos of Xenopus laevis, of three representative small nucleolar RNAs (snoRNAs), U3, U15 and U17, and of two nucleolar proteins, nucleolin and fibrillarin. The levels of the three snoRNAs in the anucleolate mutant are the same as in normal embryos, in contrast to 5S RNA and ribosomal proteins. In situ hybridization showed that, in the absence of fully organized nucleoli, the three RNAs are diffusely distributed in the nucleus and partly associated with a number of small structures. Nucleolin and fibrillarin are also present in the anucleolate embryos as in normal embryos, although there is less nucleolin mRNA in the former. The two nucleolar proteins were localized by immunofluorescence microscopy. Fibrillarin, similar to its associated U3 and U15 snoRNAs, is diffusely distributed in the anucleolate nucleus and is partly associated with small structures, probably prenucleolar bodies and pseudonucleoli. Nucleolin also appears diffusely distributed in the nucleus with some spots of higher concentration, but with a different pattern with respect to fibrillarin.

Fugu intron oversize reveals the presence of U15 snoRNA coding sequences in some introns of the ribosomal protein S3 gene.

We present here the analysis of the genomic organization of the Fugu gene coding for ribosomal protein S3 and its intron encoded U15 RNA, and compare it with the homologous human and Xenopus genes. Only two of the six Fugu S3 gene introns do not contain the U15 sequence and are in fact shorter than 100 nucleotides, as most Fugu introns. The other four introns are somewhat longer and contain sequences homologous to U15 RNA; two of these represent functional copies, as shown by microinjections of Fugu transcripts into Xenopus oocytes, whereas the other two appear to be nonfunctional pseudocopies. Thus Fugu turns out to be ideal for the study of intron encoded snoRNAs, partly because of the reduced cloning and sequencing workload, and partly because the intron length per se can be an indication of the presence of a snoRNA coding sequence.

A functional role for some Fugu introns larger than the typical short ones: the example of the gene coding for ribosomal protein S7 and snoRNA U17.

The compact genome of Fugu rubripes, with its very small introns, appears to be particularly suitable to study intron-encoded functions. We have analyzed the Fugu gene for ribosomal protein S7 (formerly S8, see Note), whose Xenopus homolog contains in its introns the coding sequences for the small nucleolar RNA U17. Except for intron length, the organization of the Fugu S7 gene is very similar to that of the Xenopus counterpart. The total length of the Fugu S7 gene is 3930 bp, compared with 12691 bp for Xenopus. This length difference is uniquely due to smaller introns. Although short, the six introns are longer than the approximately 100 bp size of most Fugu introns, as they host U17 RNA coding sequences. While four of the six U17 sequences are 'canonical', the remaining two represent diverged U17 pseudocopies. In fact, microinjection in Xenopus oocytes of in vitro synthesized Fugu transcripts containing the 'canonical' U17f sequence results in efficient production of mature U17 RNA, while injection of a transcript containing the U17 psi b sequence does not.

Structure and expression of ribosomal protein genes in Xenopus laevis.

In Xenopus laevis, as well as in other vertebrates, ribosomal proteins (r-proteins) are coded by a class of genes that share some organizational and structural features. One of these, also common to genes coding for other proteins involved in the translation apparatus synthesis and function, is the presence within their introns of sequences coding for small nucleolar RNAs. Another feature is the presence of common structures, mainly in the regions surrounding the 5' ends, involved in their coregulated expression. This is attained at various regulatory levels: transcriptional, posttranscriptional, and translational. Particular attention is given here to regulation at the translational level, which has been studied during Xenopus oogenesis and embryogenesis and also during nutritional changes of Xenopus cultured cells. This regulation, which responds to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA (ribosomal protein mRNA) engaged on polysomes. A typical 5' untranslated region characterizing all vertebrate rp-mRNAs analyzed to date is responsible for this translational behaviour: it is always short and starts with an 8-12 nucleotide polypyrimidine tract. This region binds in vitro some proteins that can represent putative trans-acting factors for this translational regulation.

The Xenopus intron-encoded U17 snoRNA is produced by exonucleolytic processing of its precursor in oocytes.

U17 is a small nucleolar RNA encoded in the introns of the Xenopus laevis gene for ribosomal protein S7 (formerly S8, see Note). To study the mechanisms involved in its in vivo processing from S7 transcripts, various in vitro synthesized RNAs embedding a U17 sequence have been microinjected into the germinal vesicle of Xenopus oocytes and their processing analysed. In particular, the Xenopus U17 gene copies a and f and a U17 gene copy from the pufferfish Fugu rubripes have been used. Information about the nature of the processing activities involved in U17 RNA maturation have been sought by injecting transcripts protected from exonucleolytic attack at their 5'-end by capping and/or lengthened at their 3'-end by polyadenylation. The results obtained indicate that U17 RNA processing is a splicing-independent event and that it is mostly or entirely due to exonucleolytic degradation at both the 5'- and 3'-ends of the precursor molecules. Moreover, it is concluded that the enzymes involved are of the processive type. It is suggested that the apparatus for U17 RNA processing is that responsible for the degradation of all excised and debranched introns. Protection from exonucleolytic attack, due to the tight structure and/or to the binding of specific proteins, would be the mechanism by which U17 RNA is produced.

Xenopus laevis ribosomal protein L22: full-length cDNA sequence and expression analysis.

A cDNA clone was isolated from a Xenopus laevis embryo library and sequenced. Primer extension experiments indicated the full-length nature of the insert and the encoded product was identified on a two dimensional gel as ribosomal protein (r-protein) L22. The 510-bp L22 cDNA sequence presents short untranslated regions and a 5'-end polypyrimidine tract found in all other vertebrate r-protein mRNA (rp mRNA) so far analyzed. Both the nucleotide (nt) and the deduced amino acid (aa) sequences have been compared with the homologous sequences from other species. The L22 nt sequence is about 70% similar to the mammalian L27a rp mRNA and about 60% homologous to the Drosophila, Tetrahymena and yeast corresponding mRNAs. The 148-aa sequence presents a higher conservation, being 90% similar to the mammalian sequence and more than 70% to the other species. Expression analysis showed that, both during X. laevis embryogenesis and in X. laevis cultured cells during growth-rate changes, L22 synthesis is translationally regulated. Therefore X. laevis L22 mRNA is a new example of the correlation between the polypyrimidine terminal tract and the translational regulation observed in other rp mRNAs.