Abstract
In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 5–7 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 5–7 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.
Highlights
Nascent transcripts undergo a co-ordinated series of modifications in the nucleus to generate mature mRNAs
We demonstrate that binding either A12 RNA or the A11G RNA motif identified by SELEX [15] to S. cerevisiae Nab2 Zn fingers 5–7 (ZnF567) induces formation of a heterotetramer containing two protein and two RNA chains, whereas ZnF567 alone is monomeric in solution
Isothermal calorimetry (ITC) indicated that both A12 and A11G RNAs bound to Nab2 Zn fingers 5–7 (ZnF567) with an ∼1:1 stoichiometry and comparable affinity, A11G RNA bound with a marginally higher affinity than A12 RNA (0.3 and 0.4 M, respectively, Supplementary Figure S1)
Summary
Nascent transcripts undergo a co-ordinated series of modifications (including 5 -capping, splicing, and 3 cleavage/polyadenylation) in the nucleus to generate mature mRNAs. Some facilitate particular functions and are generally released, other RNA-binding proteins function to compact mature transcripts to facilitate movement through NPCs (reviewed by [1]). In Saccharomyces cerevisiae, the nuclear export of mature transcripts is mediated primarily by the export factor Mex67:Mtr that binds to the mRNPs and, through interactions with NPC proteins that contain multiple phenylalanine–glycine motifs (FG-nucleoporins), facilitates their passage though the pores (reviewed by [2,3,4]). MRNP composition probably varies for different transcripts, several mRNA binding proteins, including capbinding proteins, the export factor, Mex67:Mtr, the SR protein, Npl, and Nab, are thought to be present on most, if not all, export-competent mRNPs in S. cerevisiae [5], albeit metazoan mRNPs appear to have some additional RNA-binding proteins, such as those of the exon-junction complex
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