SummaryPolyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit. Structural coherence is lost in the RNA-binding compromised Nab2-C437S mutant, which also suppresses the rat8-2 allele of RNA helicase Dbp5. Structure-guided Nab2 variants indicate that dbp5(rat8-2) suppression is more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to affinity for polyadenosine-RNA. These results indicate that, in addition to modulating polyA tail length, Nab2 has an unanticipated function associated with generating export-competent mRNPs, and that changes within fingers 5-7 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.

Poly(ADP-ribosyl)ation represents an important post-translational modification in higher eukaryotes. Several DNA repair/checkpoint proteins possess specific PAR-Binding Zinc finger (PBZ) modules critical for function. Here, we present solution structures of the two PBZ modules of APLF (Aprataxin and PNK-like factor), revealing a novel type of zinc finger. By combining in vivo PAR-binding data with NMR interaction data using PAR fragments, we suggest a structural basis for PBZ-PAR recognition.

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The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.

In the preceding article [1] we introduced a method to synthesize a library of compounds structurally based on the nonsteroidal antiinflammatory drug (NSAID) sulindac (1,[*] Dr.

The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (nucleoporin) FG repeats and Hpr1, a component of the TREX⅐THO complex that functions to link transcription and export. Using fluorescence resonance energy transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin peptide and define residues engaged in the interaction and those involved in the FXFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FXFG nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FXFG is unable to bind mono-or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.Transcripts generated by RNA polymerase II undergo a carefully orchestrated series of processing steps, including 5Ј capping, splicing, 3Ј end cleavage, and polyadenylation before being exported to the cytoplasm. The steps of mRNA biogenesis leading to export-competent ribonucleoprotein particles (mRNPs) 5 are intimately coupled (1, 2). This coordination of mRNA biogenesis is mediated by a diverse range of RNA-binding proteins and maturation enzymes (reviewed in Refs. 2 and 3). Moreover, during processing, the transcripts are under the constant surveillance of RNA quality control mechanisms that ensure that only correctly processed mRNAs are exported to the cytoplasm for translation (4 -7). From yeast to human, translocation of fully mature mRNPs to the cytoplasm through nuclear pore complex is thought to be mediated primarily by Mex67/NXF1 (also known as TAP), the primary mRNA export receptor, which forms a heterodimer with Mtr2/NXT1 (P15) that interacts directly with nuclear pore complex proteins (FG nucleoporins) that have characteristic FG sequence repeats (8 -10). Previous work has shown that, like other nuclear transport factors (reviewed in Refs. 11 and 12), the Mex67/TAP UBA domain binds specifically to the two Phe rings of the FXFG nucleoporin-binding motif (13,14). Because Mex67/NXF1 has low intrinsic affinity for mRNAs, it is recruited to the mRNP by several RNA-binding adaptors, including members of the THO⅐TREX complex that couples transcription elongation to mRNA export (2,15,16) as well as the TREX-2 complex, Yra1/ ALY and Sub2/UAP56 (reviewed in Ref. 3).Recent studies have shown that the ubiquitin pathway participates in the regulation of several key cellular functions including mRNA nuclear export (17)(18)(19). Ubiquitin is a small 76-residue protein that can be covalently linked as a mono...

In the preceding article [1] we introduced a method to synthesize a library of compounds structurally based on the nonsteroidal antiinflammatory drug (NSAID) sulindac (1,[*] Dr.