PMC:2728203 / 1335-8191 JSONTXT

{"project":"2_test","denotations":[{"id":"18190927-16141059-62517951","span":{"begin":525,"end":526},"obj":"16141059"},{"id":"18190927-17786152-62517951","span":{"begin":525,"end":526},"obj":"17786152"},{"id":"18190927-16682182-62517951","span":{"begin":525,"end":526},"obj":"16682182"},{"id":"18190927-16777596-62517951","span":{"begin":525,"end":526},"obj":"16777596"},{"id":"18190927-17289581-62517951","span":{"begin":525,"end":526},"obj":"17289581"},{"id":"18190927-16141059-62517952","span":{"begin":768,"end":769},"obj":"16141059"},{"id":"18190927-17786152-62517952","span":{"begin":768,"end":769},"obj":"17786152"},{"id":"18190927-17786152-62517953","span":{"begin":1021,"end":1022},"obj":"17786152"},{"id":"18190927-11909523-62517953","span":{"begin":1021,"end":1022},"obj":"11909523"},{"id":"18190927-16141059-62517954","span":{"begin":1471,"end":1472},"obj":"16141059"},{"id":"18190927-17786152-62517954","span":{"begin":1471,"end":1472},"obj":"17786152"},{"id":"18190927-16682182-62517954","span":{"begin":1471,"end":1472},"obj":"16682182"},{"id":"18190927-17289581-62517954","span":{"begin":1471,"end":1472},"obj":"17289581"},{"id":"18190927-17289581-62517955","span":{"begin":1704,"end":1705},"obj":"17289581"},{"id":"18190927-16783363-62517955","span":{"begin":1704,"end":1705},"obj":"16783363"},{"id":"18190927-16783364-62517955","span":{"begin":1704,"end":1705},"obj":"16783364"},{"id":"18190927-16820771-62517955","span":{"begin":1704,"end":1705},"obj":"16820771"},{"id":"18190927-17786152-62517956","span":{"begin":2094,"end":2096},"obj":"17786152"},{"id":"18190927-15933735-62517956","span":{"begin":2094,"end":2096},"obj":"15933735"},{"id":"18190927-12787776-62517956","span":{"begin":2094,"end":2096},"obj":"12787776"},{"id":"18190927-11849973-62517957","span":{"begin":2243,"end":2245},"obj":"11849973"},{"id":"18190927-17352659-62517958","span":{"begin":2444,"end":2446},"obj":"17352659"},{"id":"18190927-12210514-62517959","span":{"begin":2528,"end":2530},"obj":"12210514"},{"id":"18190927-17786152-62517960","span":{"begin":2846,"end":2848},"obj":"17786152"},{"id":"18190927-15933735-62517960","span":{"begin":2846,"end":2848},"obj":"15933735"},{"id":"18190927-15137937-62517960","span":{"begin":2846,"end":2848},"obj":"15137937"},{"id":"18190927-14718167-62517960","span":{"begin":2846,"end":2848},"obj":"14718167"},{"id":"18190927-10085285-62517961","span":{"begin":3073,"end":3075},"obj":"10085285"},{"id":"18190927-14718167-62517962","span":{"begin":3277,"end":3279},"obj":"14718167"},{"id":"18190927-16141059-62517963","span":{"begin":3414,"end":3415},"obj":"16141059"},{"id":"18190927-17786152-62517963","span":{"begin":3414,"end":3415},"obj":"17786152"},{"id":"18190927-16682182-62517963","span":{"begin":3414,"end":3415},"obj":"16682182"},{"id":"18190927-11779864-62517964","span":{"begin":3708,"end":3710},"obj":"11779864"},{"id":"18190927-11927564-62517964","span":{"begin":3708,"end":3710},"obj":"11927564"},{"id":"18190927-11927564-62517965","span":{"begin":3763,"end":3765},"obj":"11927564"},{"id":"18190927-8483450-62517966","span":{"begin":3987,"end":3989},"obj":"8483450"},{"id":"18190927-12531921-62517967","span":{"begin":4009,"end":4011},"obj":"12531921"},{"id":"18190927-12531921-62517968","span":{"begin":4122,"end":4124},"obj":"12531921"},{"id":"18190927-15137937-62517969","span":{"begin":4272,"end":4274},"obj":"15137937"},{"id":"18190927-14718167-62517969","span":{"begin":4272,"end":4274},"obj":"14718167"},{"id":"18190927-10085285-62517970","span":{"begin":4333,"end":4335},"obj":"10085285"},{"id":"18190927-11779864-62517971","span":{"begin":4700,"end":4702},"obj":"11779864"},{"id":"18190927-11927564-62517971","span":{"begin":4700,"end":4702},"obj":"11927564"},{"id":"18190927-17630287-62517971","span":{"begin":4700,"end":4702},"obj":"17630287"},{"id":"18190927-12496292-62517972","span":{"begin":4943,"end":4945},"obj":"12496292"},{"id":"18190927-12496292-62517973","span":{"begin":5355,"end":5357},"obj":"12496292"},{"id":"18190927-9488461-62517974","span":{"begin":5422,"end":5424},"obj":"9488461"},{"id":"18190927-8849456-62517975","span":{"begin":5551,"end":5554},"obj":"8849456"},{"id":"18190927-17630287-62517976","span":{"begin":5678,"end":5680},"obj":"17630287"},{"id":"18190927-8474438-62517976","span":{"begin":5678,"end":5680},"obj":"8474438"},{"id":"18190927-15208322-62517977","span":{"begin":5750,"end":5752},"obj":"15208322"},{"id":"18190927-16682182-62517978","span":{"begin":5945,"end":5947},"obj":"16682182"},{"id":"18190927-10523319-62517978","span":{"begin":5945,"end":5947},"obj":"10523319"},{"id":"18190927-10523319-62517979","span":{"begin":6092,"end":6094},"obj":"10523319"},{"id":"18190927-15208322-62517980","span":{"begin":6186,"end":6188},"obj":"15208322"}],"text":"Introduction\nGene expression depends on the production of mature mRNA transcripts that must be exported from their site of synthesis in the nucleus to the cytoplasm for translation. This complex multistep process can be conveniently subdivided into three distinct stages: generation of mature mRNP export complexes in the nucleus, their equilibration between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), and disassembly of the export complex in the cytoplasm that prevents its return to the nucleus.1–5 The generation of export-competent mRNPs is a prerequisite for nuclear export and represents the culmination of transcription, addition of a 5′-cap to the transcript, splicing out of introns, and cleavage and polyadenylation of the 3′-end.1,2 Following completion of these processing steps, the mature transcript must be translocated to the cytoplasm where it can interface with the translation machinery. Recent evidence indicates that mRNA synthesis, processing, and export are coordinated.2,6 This coordination is achieved via a large number of mRNA-binding proteins that interact with the transcript from transcription through translation and ultimately to its destruction. The export of mature mRNPs through NPCs is mediated primarily by an evolutionarily conserved heterodimer, termed Mex67:Mtr2 in yeast, via its transient interactions with phenylalanine glycine repeat-containing proteins of the nuclear pore called FG nucleoporins.1–3,5 When the export complex reaches the cytoplasm, the RNA helicase, Dbp5, and Gle1 mediate its disassembly, and this process has been proposed to function as a molecular ratchet that prevents the return of the mRNP to the nucleus.5,7–9\nCells require an efficient mechanism to establish when mRNA is mature and competent for export. Thus, there are a number of cellular quality-control mechanisms that work actively to destroy improperly processed transcripts and preferentially export correctly processed mRNA to complement the cellular factors that assure coordinated production and export of correct mRNA transcripts.2,10,11 The nuclear exosome, a complex of 3′–5′ riboexonucleases, can detect and destroy aberrant or incorrectly processed transcripts within the nucleus.12 Several additional quality-control mechanisms function posttranscriptionally and include the nonsense-mediated decay pathway, which recognizes transcripts with defects such as premature stop codons,13 and the nonstop decay pathway, which recognizes transcripts lacking a stop codon.14 Recognition of transcripts by any of these pathways leads to their destruction. In addition to these “search-and-destroy” quality-control mechanisms in the nucleus and the cytoplasm, recent work has uncovered evidence in Saccharomyces cerevisiae that surveillance of mRNA can also occur at the nuclear pore.2,10,15,16 These studies suggest that specific interactions between mRNA-binding proteins associated with a given transcript and the evolutionarily conserved myosin-like protein 1 (Mlp1), located at the inner nuclear basket of the NPC,17 can influence nuclear export of that transcript. For example, if introns are retained in a transcript, interactions between splicing factors and Mlp1 lead to retention of the transcript in the nucleus.16\nHeterogeneous nuclear ribonucleoproteins (hnRNPs) are important in orchestrating the different steps of the gene expression pathway.1–3 One important hnRNP function is to sort export-competent mRNPs and concentrate them in the vicinity of NPCs to facilitate export. Nuclear abundant poly(A) RNA-binding protein (Nab2) is an essential multidomain hnRNP that is required for the nuclear export of poly(A) mRNA in S. cerevisiae.18,19 Nab2 is also involved in poly(A) tail length control19 and has been proposed to provide a link between the termination of polyadenylation and mRNA nuclear export. Nab2 interacts with Mlp1, which is constructed from a long α-helical coiled coil and a globular C-terminal domain20 that binds to Nab2.21 Overexpression of Mlp1 or its C-terminal domain causes retention of both Nab2 and poly(A)+ RNA in the nucleus.21 Mlp1 is itself attached to the nucleoplasmic face of NPCs and, hence, is thought to facilitate coordination of several steps in mRNA metabolism,15,16 albeit the Mlp proteins are not essential for cell growth.17 Because it both recognizes the presence of a poly(A) tail and interacts with Mlp1, Nab2 is an attractive candidate to participate in the final step of the nuclear portion of the gene expression pathway.\nNab2 shuttles between the nucleus and the cytoplasm and is thought to interact with transcripts following 3′-end cleavage and addition of the poly(A) tail.18,19,22 Thus, Nab2 and other shuttling mRNA-binding proteins could serve as positive markers to target mature transcripts for preferential export from the nucleus. The Nab2 protein (Fig. 1a) has a modular structure based on four functional domains:23 a unique N-terminal domain of approximately 100 residues, a glutamine-rich linker, an RGG domain, and a Zn-finger domain. Deletion analysis indicates that the N-terminal domain is necessary for the nuclear export of both poly(A) RNA and Nab2 in vivo, consistent with a model in which Nab2 associates with mRNA in the nucleus and in which the N-terminal domain facilitates export of both poly(A) mRNA and Nab2.23 The RGG domain, which contains the nuclear localization sequence24 recognized by the nuclear import factor Kap104 (also known as transportin or Kapβ2), is required for the nuclear import of Nab225 to recycle it for another round of mRNA export. The Zn-finger domain is critical for Nab2 binding to polyadenosine mRNA.22,26\nThe N-terminal domain of Nab2 interacts with the export factor, Gfd1,27 a protein that may be involved in the terminal step of the export pathway involving disassembly of the export complex in the cytoplasm by the RNA helicase, Dbp5, and the export factor, Gle1.3,28 Gfd1 is a high-copy suppressor of the rat8-2 allele of DBP5 and binds to both Gle1 and the cytoplasmically oriented nuclear pore protein, Nup42,28 and, thus, has the potential to link Nab2 to both Gle1 and the cytoplasmic face of the NPC.27\nHere, we show that the N-terminal domain of Nab2 interacts with the C-terminal domain of Mlp1. Our studies demonstrate that the N-terminal domain of Nab2 is both necessary and sufficient to mediate interaction with Mlp1. We have determined the atomic resolution structure of the Nab2 N-terminal domain, using both NMR and X-ray crystallography, and show that it is analogous to the PWI fold found in several RNA-binding proteins. We have identified a putative protein:protein interaction site containing a hydrophobic surface patch centered on Phe73 that mutagenesis indicates is involved in the interaction between Nab2 and Mlp1 but not in the interaction with Gfd1."}