Evolutionarily Conserved Polyadenosine RNA Binding Protein Nab2 Cooperates with Splicing Machinery To Regulate the Fate of Pre-mRNA

Abstract

g Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate posttranscriptional 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 study 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 a splicing factor, MUD2, and 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/U2AF 65 . 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. Gene expression is temporally and spatially regulated to produce a precise protein expression profile that dictates the function of each cell. Although much of this control occurs at the level of transcription, both co-and posttranscriptional events also play key regulatory roles. Newly synthesized mRNAs undergo numerous processing events, including 5= capping, splicing, 3=-end processing, and export to the cytoplasm (1, 2). Ensuring the synchrony of mRNA biogenesis requires RNA binding proteins that not only perform the processing tasks but also couple the events to ensure that only properly processed mRNAs are available for translation in the cytoplasm (3).Key processing events that must be coordinated include splicing and 3=-end processing. Although steps in mRNA processing are often depicted and studied as separate events, there is a growing body of evidence that these processing events are intimately coupled to one another (2). For example, splicing and 3=-end processing are coupled in humans as mutations in splice site and polyadenylation consensus sequences mutually disrupt both splicing and polyadenylation (4-6). In addition, a number of splicing factors copurify with the 3=-end processing complex (7-10). For example, there is evidence that the splicing factor U2AF2/ U2AF 65 functions as a bridge between the U2 snRNP and the 3=-end processing machinery (11,12). Given the extensive coupling between RNA processing steps, it is important to consider the consequences when one step of the process is disrupted in vivo. Cells have developed numerous overlapping mechanisms to ensure that faulty mRNA transcripts are not...

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA

“…Previous studies performed in budding yeast provide evidence that Nab2 is involved in the generation of export-competent messenger ribonucleoproteins and retention of improperly processed RNAs (26,29,30). Consistent with a similar function for ZC3H14, ATP5G1 pre-mRNA levels increase in the cytoplasm upon ZC3H14 knockdown (Fig.…”

“…Previous work in orthologous systems demonstrates that ZC3H14 has specificity for polyadenosine RNA (15) and plays a role in poly(A) tail length control (24), mRNA export (25), target transcript stability (28), and RNA quality control in the nucleus (29,30); however, we cannot rule out the possibility that ZC3H14 affects ATP5G1 transcription. To test for transcriptional changes to ATP5G1, -2, or -3 upon knockdown of ZC3H14, we performed qRT-PCR analyses with transcriptspecific primers to amplify the pre-mRNAs for each of the three subunit transcripts (see Fig.…”

“…Knockdown of ZC3H14 Decreases ATP5G1 mRNA LevelsPrevious studies of ZC3H14 orthologs in model organisms suggest a spectrum of post-transcriptional functions (24,25,28) that may influence the fate of bound target transcripts (29). To examine the function of ZC3H14 in human cells, we sought to identify candidate target mRNA transcripts via an unbiased genome-wide approach.…”

“…Extensive work on the Saccharomyces cerevisiae ortholog of ZC3H14, Nab2 (22,23), reveals that this essential protein plays roles in poly(A) tail length control and mRNA export (24 -27), target transcript stability (28), and RNA quality control in the nucleus (29,30). Genetic analyses performed in Drosophila melanogaster reveal a conserved function of Drosophila Nab2 (dNab2) in post-transcriptional processing, specifically in neurons (31), consistent with the brain dysfunction present in patients.…”

Structure–function relationships in the Nab2 polyadenosine‐RNA binding Zn finger protein family

“…Sc Nab2 is then recycled back to the nucleus through nuclear pores using the transport factor, karyopherin β2 (also termed Kap104 or transportin) . In addition, Sc Nab2 shows genetic interactions with the splicing machinery and also appears to function in mRNA quality control and Pol‐III transcription …”