The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA.

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo We extend this analysis to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65 Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.

Poly(A) tail-mediated gene regulation by opposing roles of Nab2 and Pab2 nuclear poly(A)-binding proteins in pre-mRNA decay.

The 3' end of most eukaryotic transcripts is decorated by poly(A)-binding proteins (PABPs), which influence the fate of mRNAs throughout gene expression. However, despite the fact that multiple PABPs coexist in the nuclei of most eukaryotes, how functional interplay between these nuclear PABPs controls gene expression remains unclear. By characterizing the ortholog of the Nab2/ZC3H14 zinc finger PABP in Schizosaccharomyces pombe, we show here that the two major fission yeast nuclear PABPs, Pab2 and Nab2, have opposing roles in posttranscriptional gene regulation. Notably, we find that Nab2 functions in gene-specific regulation in a manner opposite to that of Pab2. By studying the ribosomal-protein-coding gene rpl30-2, which is negatively regulated by Pab2 via a nuclear pre-mRNA decay pathway that depends on the nuclear exosome subunit Rrp6, we show that Nab2 promotes rpl30-2 expression by acting at the level of the unspliced pre-mRNA. Our data support a model in which Nab2 impedes Pab2/Rrp6-mediated decay by competing with Pab2 for polyadenylated transcripts in the nucleus. The opposing roles of Pab2 and Nab2 reveal that interplay between nuclear PABPs can influence gene regulation.

Structural basis for polyadenosine-RNA binding by Nab2 Zn fingers and its function in mRNA nuclear export.

Polyadenylation 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.

The long and the short of it: the role of the zinc finger polyadenosine RNA binding protein, Nab2, in control of poly(A) tail length.

In eukaryotic cells, addition of poly(A) tails to transcripts by 3'-end processing/polyadenylation machinery is a critical step in gene expression. The length of the poly(A) tail influences the stability, nuclear export and translation of mRNA transcripts. Control of poly(A) tail length is thus an important mechanism to regulate the abundance and ultimate translation of transcripts. Understanding the global regulation of poly(A) tail length will require dissecting the contributions of enzymes, regulatory factors, and poly(A) binding proteins (Pabs) that all cooperate to regulate polyadenylation. A recent addition to the Pab family is the CCCH-type zinc finger class of Pabs that includes S. cerevisiae Nab2 and its human counterpart, ZC3H14. In S. cerevisiae, Nab2 is an essential nuclear Pab implicated in both poly(A) RNA export from the nucleus and control of poly(A) tail length. Consistent with an important role in regulation of poly(A) tail length, depletion of Nab2 from yeast cells results in hyperadenylation of poly(A) RNA. In this review, we focus on the role of Nab2 in poly(A) tail length control and speculate on potential mechanisms by which Nab2 could regulate poly(A) tail length based on reported physical and genetic interactions. We present models, illustrating how Nab2 could regulate poly(A) tail length by limiting polyadenylation and/or enhancing trimming. Given that mutation of the gene encoding the human Nab2 homologue, ZC3H14, causes a form of autosomal recessive intellectual disability, we also speculate on how mutations in a gene encoding a ubiquitously expressed Pab lead specifically to neurological defects. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.