Abstract
Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3′-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length.
Highlights
Following transcription by RNA polymerase II in the nucleus, mRNA transcripts must be spliced and polyadenylated, exported to the cytoplasm, and perhaps even transported to a distant site of translation [1]
These data suggest that if the RNA binding affinity of a Dbp5 substrate, such as nuclear poly(A)-binding protein 2 (Nab2), is decreased, as in Nab2-C437S, a partially functional mutant Dbp5 protein may retain sufficient remodeling activity to remove the weakly bound Nab2-C437S protein from the mRNA transcript. This model implies that other Nab2 mutants that weaken the interaction of Nab2 with polyadenosine RNA may suppress the temperature-sensitive phenotype of the rat8-2 mutant
To determine which of the seven zinc fingers in Nab2 are important for RNA binding, we generated point mutants that result in cysteine to alanine amino acid changes in the first cysteine of each of the seven zinc fingers (Fig. 1A), and we tested whether these mutants could suppress the temperature-sensitive growth phenotype of the rat8-2 mutant at 32 °C
Summary
Cells that expressed Nab RNA-binding mutants showed an increase in poly(A) tail length but no nuclear accumulation of poly(A)RNA suggesting that Nab binding to polyadenosine RNA is required for the modulation of poly(A) tail length. These nab variant alleles genetically interacted with mutant alleles of the mRNA 3Ј-end processing machinery. These results provide critical insight into the molecular mechanism underlying polyadenosine RNA recognition by CCCH zinc fingers as well as the means by which Pabs can post-transcriptionally regulate gene expression
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