Functional Significance of the Interaction between the mRNA-binding Protein, Nab2, and the Nuclear Pore-associated Protein, Mlp1, in mRNA Export

Abstract

Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CTMlp1: residues 1490 -1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe 73 that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586 -1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.Production of mature mRNA for translation is a complex multistep process involving an array of RNA-binding proteins (1-3). From its transcriptional inception in the nucleus, pre-mRNA that emerges from RNA polymerase II must be bound by processing factors for 5Ј-end capping, splicing, and 3Ј-end cleavage and polyadenylation to reach maturation (4). Alongside these processing events, maturing mRNA must be co-transcriptionally loaded with heterogeneous nuclear ribonucleoproteins (hnRNPs), 2 mRNA export adaptors, and an mRNA export receptor to facilitate its active export from the nucleus to the cytoplasm through nuclear pore complexes (NPCs), large proteinaceous channels that perforate the nuclear membrane and mediate nucleocytoplasmic transport (1-3, 5). There are a number of nuclear quality controls that safeguard against improperly processed mRNA being exported and translated into deleterious proteins (6, 7). In particular, this quality control machinery likely monitors the state of processed mRNA by checking for the presence or absence of mRNA processing and export factors.One of the key players in mRNA export is the conserved heterodimeric mRNA export receptor, Mex67/Mtr2 (NXF1/ NXT1 or TAP/p15 in metazoans), which plays an essential role in bulk mRNA export in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster...

Quantitative proteomic analysis reveals concurrent RNA–protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae

“…Strikingly, ;50% (114/220) of the RNA-dependent binders we identified were already known to be RBPs (enrichment P-value 2 3 10 À35 ), which is a considerable improvement over previous approaches. The RBP Nab2 is involved in the end processing, polyadenylation, and export of poly(A) mRNA from the nucleus; we see both known and novel RNAdependent interactions with Nab2 that are consistent with the existing model (Hector et al 2002;Tran et al 2007;Fasken et al 2008;Iglesias and Stutz 2008;Tutucci and Stutz 2011). However, our data provide new insight into the temporal program of Nab2 binding based on evidence for concurrent binding with RBPs involved in transcription, splicing, and translation.…”

“…Therefore, there is a >95% chance that Yhr097c cross-links with higher efficiency than the negative controls. Nab2 is involved in the end processing, polyadenylation, and export of poly(A) mRNA from the nucleus (Green et al 2002;Hector et al 2002;Fasken et al 2008;Iglesias and Stutz 2008;Tutucci and Stutz 2011). It is generally believed that Nab2 binds to mRNAs during their end cleavage and polyadenylation and is removed immediately following their nuclear export (Lee and Aitchison 1999;Tran et al 2007).…”

The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway

“…This is of interest since Mex67 is the Nxf1 yeast ortholog and has been shown to be a major mRNA export factor (Segref et al 1997;Katahira et al 1999). Mlp1 has also been shown to interact with Nab2, a poly(A)-binding protein that also interacts with Mex67 (Green et al 2003a;Fasken et al 2008). Currently, no definitive mammalian ortholog for Nab2 has been identified.…”

Purification of Nuclear Poly(A)-binding Protein Nab2 Reveals Association with the Yeast Transcriptome and a Messenger Ribonucleoprotein Core Structure

“…Nab2 is mainly localized in the nucleus (7,8) and interacts with the poly(A)-binding protein (Pab1) to affect poly(A) tail length (9). Moreover, Nab2 interacts with Mlp1, a nuclear pore complex-associated protein at the nuclear basket involved in mRNP quality control (10), with Gfd1 and Gle1, factors involved in mRNA export (11), and with the Sac3-Thp1 complex, which plays a role in coupling transcription with mRNA metabolism (12). When the pools of mRNAs immuneprecipitated by Npl3, Nab2, and Nab4 were determined in microarrays and compared, it was suggested that distinct sets of mRNAs are bound to each of these proteins (13).…”