ABSTRACT
The TREX-2 complex of eukaryotes is responsible for the export of a wide range of mRNAs from the nucleus to the cytoplasm. Previously, we showed that a subunit of the D. melanogaster TREX-2 complex, the PCID2 protein, has a domain that specifically interacts with RNA. However, it remains unknown whether other components of the complex are involved in interaction with and recognition of the target mRNA. In the present study, we determined the role of Xmas-2, the core structural subunit of the complex, in the specific recognition of ras2 mRNA fragments. In this work, we showed that Xmas-2 interacts with ras2 mRNA independently of other subunits of the complex. We showed that RNA-binding domains are located in both the N-terminal domain and the C-terminal domain of Xmas-2. However, the interaction of the protein with ras2 mRNA fragments is independent of RNA sequence and structure and is nonspecific. Thus, the Xmas-2 subunit is not involved in the recognition of specific RNA sequences by the complex.
Subject(s)
RNA, Messenger , Animals , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Protein Binding , ras Proteins/metabolism , ras Proteins/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/chemistryABSTRACT
The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.
Subject(s)
Drosophila melanogaster , Histones , Animals , Active Transport, Cell Nucleus , Histones/metabolism , Drosophila melanogaster/genetics , RNA, Messenger/genetics , Nuclear Proteins/metabolism , Cell Nucleus/metabolismABSTRACT
The TREX-2 protein complex is the key complex involved in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a joint protein complex of TREX-2 with ORC was isolated in D. melanogaster. It was shown that the interaction of TREX-2 with ORC is necessary for efficient mRNA export from the nucleus to the cytoplasm. In this work, we showed that the TREX-2-ORC joint complex is also formed in human cells.
Subject(s)
Drosophila melanogaster , Nuclear Proteins , Animals , Humans , Active Transport, Cell Nucleus , Cell Nucleus/genetics , Cell Nucleus/metabolism , Drosophila melanogaster/metabolism , Nuclear Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Humans , Cell Nucleus/metabolism , Drosophila melanogaster/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Nuclear Proteins/metabolism , RNA, Messenger/genetics , Transcription Factors/metabolismABSTRACT
The TREX-2 protein complex is the key participant in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a protein complex of D. melanogaster consisting of TREX-2 and ORC complexes was purified. It was shown that, in the TREX-2-ORC complex, the Xmas-2 protein, which is the platform for TREX-2 assembly, interacts with the Orc3 protein. The aim of this work was to investigate what regions of the Xmas-2 amino acid sequence are involved in the interaction with Orc3. It was shown that the interaction of Xmas-2 with Orc3 requires a C-terminal region of Xmas-2 located downstream of the CID domain.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Drosophila melanogaster/genetics , Protein Interaction Domains and Motifs , RNA Transport , RNA, Messenger/genetics , Sequence Homology, Amino AcidABSTRACT
TREX-2 complex is responsible for general mRNA export from nucleus to cytoplasm in eukaryote. The main protein of TREX-2 complex of D. melanogaster is protein Xmas-2. Its homologues in yeast and humans are Sac3 and GANP proteins, respectively. All three proteins contain the highly conserved domain Sac3-GANP, which is essential for interaction of TREX-2 complex with mRNA and another protein of the complex, PCID2. We identified two Xmas-2 homologues in D. melanogaster using the Sac3-GANP family domain sequence. These proteins have a common domain responsible for interaction with the PCID2 protein and RNA and are present in other eukaryotes. The function of these proteins is unknown. However, on the basis of their structural organization, we can assume that they interact with nucleic acids.