ABSTRACT
The proteins Mago and Y14 are evolutionarily conserved binding partners. Y14 is a component of the exon-exon junction complex (EJC), deposited by the spliceosome upstream of messenger RNA (mRNA) exon-exon junctions. The EJC is implicated in post-splicing events such as mRNA nuclear export and nonsense-mediated mRNA decay. Drosophila Mago is essential for the localization of oskar mRNA to the posterior pole of the oocyte, but the functional role of Mago in other species is unknown. We show that Mago is a bona fide component of the EJC. Like Y14, Mago escorts spliced mRNAs to the cytoplasm, providing a direct functional link between splicing and the downstream process of mRNA localization. Mago/Y14 heterodimers are essential in cultured Drosophila cells. Taken together, these results suggest that, in addition to its specialized function in mRNA localization, Mago plays an essential role in other steps of mRNA metabolism.
Subject(s)
Drosophila Proteins , Nuclear Proteins/metabolism , RNA Splicing/genetics , RNA, Messenger/metabolism , Animals , Biological Transport , Cell Division , Cell Line , Cell Nucleus Structures/metabolism , Cytoplasm/metabolism , DNA/genetics , Dimerization , Drosophila/cytology , Drosophila/genetics , Drosophila/metabolism , Evolution, Molecular , Exons/genetics , HeLa Cells , Humans , Male , Nuclear Proteins/genetics , Polymerase Chain Reaction , Protein Binding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Testis , Transfection , XenopusABSTRACT
Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Nucleus/metabolism , Drosophila Proteins/metabolism , RNA Helicases/metabolism , RNA, Messenger/metabolism , Active Transport, Cell Nucleus , Adenosine Triphosphatases/genetics , Animals , Cells, Cultured , Cloning, Molecular , DEAD-box RNA Helicases , DNA, Complementary , Drosophila melanogaster , HeLa Cells , Heat-Shock Response , Humans , Protein Biosynthesis , RNA Helicases/genetics , RNA Splicing , RNA, Antisense , RNA, Small InterferingABSTRACT
We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. When assembled in vitro, this so-called 'exon-exon junction complex' (EJC) contains at least five proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to significant evolution upon mRNA export to the cytoplasm.
Subject(s)
Antigens, Nuclear , Exons , Nuclear Matrix-Associated Proteins , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins , Animals , Binding Sites , Cloning, Molecular , DNA-Binding Proteins/metabolism , Escherichia coli , Humans , In Vitro Techniques , Models, Genetic , Nuclear Proteins/metabolism , Oocytes/physiology , RNA Precursors/metabolism , Recombinant Proteins/metabolism , Xenopus laevisABSTRACT
Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.
Subject(s)
Antigens, Nuclear , Exons , Nuclear Matrix-Associated Proteins , RNA, Messenger/genetics , Ribonucleoproteins , Spliceosomes/metabolism , Animals , Cell Nucleus/physiology , Cytoplasm/physiology , DNA-Binding Proteins/metabolism , Female , Half-Life , HeLa Cells , Humans , Nuclear Proteins/metabolism , Oocytes/physiology , Protein Biosynthesis , RNA Splicing , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease H , Xenopus laevisABSTRACT
Activating mutations of the RET oncogene cause the inheritance of multiple endocrine neoplasia type 2 (MEN2). The RET pre-mRNA is spliced into several transcripts coding for multiple isoforms, including Ret9 and Ret51. When harboring activating mutations in the cytoplasmic region, the Ret51 protein displays a higher in vitro transforming efficiency as compared to the corresponding Ret9 isoform. We investigated whether a more transforming isoform was preferentially expressed in MEN2 tumors as compared to normal tissues or sporadic pheochromocytomas. By quantitative RNases protection assays, we measured the absolute abundance of the 3' splice variants in pheochromocytomas and in normal tissues. The proportion of RET51 transcripts was highly dispersed between tumors and normal tissues. In familial tumors the proportion of RET51 transcripts was significantly larger (48.1%) than in sporadic tumors (36.75%). This result suggests that the preferential expression of the Ret51 protein isoform, even though moderate, is a growth advantage for MEN2 tumors.
Subject(s)
Adrenal Gland Neoplasms/metabolism , Drosophila Proteins , Multiple Endocrine Neoplasia Type 2a/metabolism , Multiple Endocrine Neoplasia Type 2b/metabolism , Pheochromocytoma/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Adrenal Gland Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Germ-Line Mutation , Humans , Isomerism , Pheochromocytoma/genetics , Proto-Oncogene Proteins c-ret , RNA Probes , RNA SplicingABSTRACT
We provide direct evidence that pre-mRNA splicing alters mRNP protein composition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon-exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SRm160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation showed that a subset of these proteins remain associated with mRNA after its release from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon-exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.
Subject(s)
Antigens, Nuclear , Exons , Introns , Nuclear Matrix-Associated Proteins , RNA Precursors/metabolism , RNA Splicing , Ribonucleoproteins/genetics , Base Sequence , Cell Nucleus/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , RNA Precursors/chemical synthesis , RNA Precursors/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U5 Small Nuclear/isolation & purification , Ribonucleoprotein, U5 Small Nuclear/metabolism , Ribonucleoproteins/metabolism , Spliceosomes/metabolismABSTRACT
Pheochromocytomas are tumors originating from chromaffin cells, the large majority of which are sporadic neoplasms. The genetic and molecular events determining their tumorigenesis continue to remain unknown. On the other hand, RET germ-line mutations cause the inheritance of familial tumors in multiple endocrine neoplasia (MEN)-2 diseases, which account for a minority of pheochromocytomas. We investigated the expression of the RET gene in 14 sporadic tumors harboring no activating mutations. A subset of highly RET-expressing tumors (50%) could be distinguished. They showed RET transcript, protein amounts as well as Ret-associated phosphotyrosine levels similar to those measured in MEN-2A-associated pheochromocytomas. We also determined the GDNF and GDNF family receptor alpha (GFRalpha)-1 transcript levels in tumors and in normal tissues. Whereas the GFRalpha-1 transcripts were detected at similar levels in normal tissues and in tumors, GDNF was frequently found expressed in sporadic tumors at levels several times higher than in controls. These results led us to propose the existence of an autocrine or paracrine loop leading to chronic stimulation of the Ret signaling pathway, which could participate in the pathogenesis of a number of sporadic pheochromocytomas.
Subject(s)
Adrenal Gland Neoplasms/metabolism , Drosophila Proteins , Nerve Growth Factors , Pheochromocytoma/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Adrenal Gland Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Glial Cell Line-Derived Neurotrophic Factor , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Pheochromocytoma/genetics , Phosphorylation , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Signal Transduction , TyrosineABSTRACT
Alternative splicing of human tyrosine hydroxylase (TH) pre-mRNA produces four mRNAs leading to four different TH isoforms and is thought to have important regulatory functions. We show that the diversity of TH mRNAs is greater than previously described in the autonomous nervous system: New splice junctions corresponding to the skipping of exon 3 were identified by amplification of cDNA synthesized from pheochromocytoma RNA. In all cases the reading frame was maintained. These species were assayed by RNase protection experiments; their abundance (4-6%) was comparable to that of the previously identified human TH-3 and -4 species in normal adrenal medulla. However, higher levels (11-34%) of these species were found in adrenal medullas of patients suffering from progressive supranuclear palsy. Whether such changes are specific to the disease or the consequences of the stress associated with this severe neurodegeneration remains to be established.
Subject(s)
Adrenal Medulla/enzymology , Supranuclear Palsy, Progressive/enzymology , Supranuclear Palsy, Progressive/genetics , Tyrosine 3-Monooxygenase/genetics , Alternative Splicing/physiology , Base Sequence , Exons/genetics , Gene Expression/physiology , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA Precursors/analysis , RNA Precursors/genetics , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RibonucleasesABSTRACT
Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain. A recombinant protein was produced in E. coli. Its in vitro specific activity was similar to that of the previously identified TH protein.
