Functional characterization of mRNA export receptor TAP-interacting proteins ASF/SF2 and DDX3

• Main Authors: Ming-Chih Lai, 賴銘志
• Other Authors: Woan-Yuh Tarn
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/25586892446720233695

博士 === 國防醫學院 === 生命科學研究所 === 96 === Nuclear export of mRNA is an important step for eukaryotic gene expression and is tightly linked to transcription and nuclear pre-mRNA processing, including splicing and polyadenylation, and subsequent maturation in the cytoplasm. TAP is the major mRNA export receptor that targets messenger ribonucleoproteins (mRNPs) to the nuclear pore complexes (NPCs). TAP and its associated proteins may act to coordinate different steps of gene expression. To better understand the TAP-mediated mRNA export pathway, we searched for TAP-associated proteins and identified several candidates including NPC proteins, RNA binding proteins, RNA helicases and ribosomal proteins. Among those, we characterized both serine/arginine (SR) dipeptide-rich protein ASF and DEAD-box RNA helicase DDX3. In addition to a major role in pre-mRNA splicing, a subset of nucleocytoplasmic shuttling SR proteins can serve as adaptors for mRNA export. SR proteins are dynamically phosphorylated in their RS domain, and differential phosphorylation modulates their splicing activity and subcellular localization. We verified the interaction of TAP with shuttling SR protein ASF. Overexpressed ASF can recruit TAP to SR protein-enriched nuclear speckles. We further found that TAP prefered to associate with hypophosphorylated SR proteins, including ASF, and that hypophosphorylated ASF was present in mature mRNPs. Therefore, dephosphorylation of SR proteins may occur during mRNA maturation and recruit TAP to mRNPs for nuclear export (J. Biol. Chem. 279, 31745-31749; 2004). DDX3 and its homologs have been implicated in various aspects of mRNA metabolism. We confirmed both in vivo and in vitro interactions between TAP and DDX3. Previous reports have indicated that nuclear export of DDX3 is mediated by importin- family protein CRM1. Thus, our finding suggests an additional pathway for the DDX3 export, which involves TAP. In addition, we observed that DDX3 was recruited into cytoplasmic stress granules (SGs) under cell stress conditions, which suggests a role of DDX3 in translational control. In accordance with this observation, DDX3 associated with translation initiation complexes. Our results further demonstrated that DDX3 was not critical for general mRNA translation, but may participate in the translation of mRNAs containing a long or structured 5’ untranslated region (UTR). Moreover, the RNA helicase activity of DDX3 appeared to be required for its function in translational control. We suggest that DDX3 facilitates translation by resolving secondary structures of the 5’ UTR in mRNAs during ribosome scanning. Taken together, our findings indicate that DDX3 associates with export mRNPs via its interaction with TAP in the nucleus and subsequently participates in the translational control of specific mRNAs after export.