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/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

Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders

A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies

Links between mRNA splicing, mRNA quality control, and intellectual disability: DOI: 10.14800/rd.1448

In recent years, the impairment of RNA binding proteins that play key roles in the post-transcriptional regulation of gene expression has been linked to numerous neurological diseases. These RNA binding proteins perform critical mRNA processing steps in the nucleus, including splicing, polyadenylation, and export. In many cases, these RNA binding proteins are ubiquitously expressed raising key questions about why only brain function is impaired. Recently, mutations in the ZC3H14 gene, encoding an evolutionarily conserved, polyadenosine RNA binding protein, have been linked to a nonsyndromic form of autosomal recessive intellectual disability. Thus far, research on ZC3H14 and its Nab2 orthologs in budding yeast and Drosophila reveals that ZC3H14/Nab2 is important for mRNA processing and neuronal patterning. Two recent studies now provide evidence that ZC3H14/Nab2 may function in the quality control of mRNA splicing and export and could help to explain the molecular defects that cause neuronal dysfunction and lead to an inherited form of intellectual disability. These studies on ZC3H14/Nab2 reveal new clues to the puzzle of why loss of the ubiquitously expressed ZC3H14 protein specifically affects neurons

Human RALY RNA binding protein suppresses <i>air1/2</i> thermosensitive growth.

<p>(<b>A</b>) Schematic comparison of Nab3 and human RALY (hRALY) Isoform 1 (GenBank accession number Q9UKM9) RNA binding protein showing percentage sequence identity between RNA recognition domain (RRM) and C-terminal domains. Residue positions are depicted above and below proteins. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s006" target="_blank">S6 Fig.</a><b>(B)</b> Alignment of hRALY and Nab3 RRMs showing that the hRALY RRM has 31% identity (identical residues shaded in gray) and a similar predicted β1α1β2β3 α2β4 secondary structure (99.8% confidence; α-helices in green; β-sheets in blue) to the Nab3 RRM. Nab3 RRM RNA-binding residues R331 and F333 that are conserved in the hRALY RRM as R22 and F24 are boxed. RRM consensus motifs RNP1 and RNP2 are underlined. Alignment and secondary structure prediction of hRALY RRM based on Nab3 RRM crystal structure (PDB ID: 2XNQ [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref063" target="_blank">63</a>]) was generated by Phyre2 server [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref064" target="_blank">64</a>] (<b>C</b>) <i>hRALY</i> suppresses the <i>air1-C178R air2</i>Δ thermosensitive growth at 30°C. The <i>air1-C178R air2</i>Δ cells containing vector, <i>NAB3</i>, <i>nab3–11</i> or <i>hRALY 2μ URA3</i> plasmid were spotted and grown at indicated temperatures. <b>(D)</b><i>hRALY</i> RRM mutants <i>hRALY-R22A</i> and <i>hRALY-F24A</i> do not suppress the <i>air1-C178R air2</i>Δ thermosensitive growth at 30°C. The <i>air1-C178R air2</i>Δ cells containing vector, <i>AIR1</i>, <i>NAB3</i>, <i>nab3–11</i>, <i>hRALY</i>, <i>hRALY-R22A</i> or <i>hRALY-F24A 2μ URA3</i> plasmid were spotted and grown at indicated temperatures. <b>(E)</b> hRALY-R22A and hRALY-F24A RRM mutant proteins are expressed in <i>air1-C178R air2</i> Δ cells but not to the same level as hRALY. Lysates of <i>air1-C178R air2∆</i> cells expressing Myc-tagged hRALY, hRALY-R22A or hRALY-F24A at 30°C were analyzed by immunoblotting to detect Myc-tagged proteins and 3-phosphoglycerate kinase (Pgk1) as a loading control. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s007" target="_blank">S7A</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s007" target="_blank">S7C Fig.</a></p

Nab3 Facilitates the Function of the TRAMP Complex in RNA Processing via Recruitment of Rrp6 Independent of Nrd1

<div><p>Non-coding RNAs (ncRNAs) play critical roles in gene regulation. In eukaryotic cells, ncRNAs are processed and/or degraded by the nuclear exosome, a ribonuclease complex containing catalytic subunits Dis3 and Rrp6. The TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex is a critical exosome cofactor in budding yeast that stimulates the exosome to process/degrade ncRNAs and human TRAMP components have recently been identified. Importantly, mutations in exosome and exosome cofactor genes cause neurodegenerative disease. How the TRAMP complex interacts with other exosome cofactors to orchestrate regulation of the exosome is an open question. To identify novel interactions of the TRAMP exosome cofactor, we performed a high copy suppressor screen of a thermosensitive <i>air1/2</i> TRAMP mutant. Here, we report that the Nab3 RNA-binding protein of the Nrd1-Nab3-Sen1 (NNS) complex is a potent suppressor of TRAMP mutants. Unlike Nab3, Nrd1 and Sen1 do not suppress TRAMP mutants and Nrd1 binding is not required for Nab3-mediated suppression of TRAMP suggesting an independent role for Nab3. Critically, Nab3 decreases ncRNA levels in TRAMP mutants, Nab3-mediated suppression of <i>air1/2</i> cells requires the nuclear exosome component, Rrp6, and Nab3 directly binds Rrp6. We extend this analysis to identify a human RNA binding protein, RALY, which shares identity with Nab3 and can suppress TRAMP mutants. These results suggest that Nab3 facilitates TRAMP function by recruiting Rrp6 to ncRNAs for processing/degradation independent of Nrd1. The data raise the intriguing possibility that Nab3 and Nrd1 can function independently to recruit Rrp6 to ncRNA targets, providing combinatorial flexibility in RNA processing.</p></div

Nab3 RNA recognition motif is essential for suppression of <i>air1/2</i> thermosensitive growth.

<p>(<b>A</b>) NMR structure of Nab3 RRM in complex with UCUU RNA, which highlights key Nab3 RRM residues that contact the RNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref041" target="_blank">41</a>]. The Nab3 RRM-RNA NMR structure (PDB ID: 2L41) shows that the Nab3 RRM forms a four-stranded β-sheet packed against two α–helices [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref041" target="_blank">41</a>]. The Nab3 RRM (green; Nab3 residues 321–415) interacts with the U₁C₂U₃U₄ RNA oligonucleotide (black). Nab3 RRM β-strand residues, Arg331 (red), Phe333 (yellow), and Ser399 (orange), make specific contacts with the C₂ nucleotide. Ser399 (orange) also contacts the U₃ nucleotide. The Nab3 RRM residues, Phe371 (blue) and Pro374 (brown), mutated in the nab3–11 (nab3-F371L-P374L) RRM mutant, are also highlighted. The Nab3 RRM-RNA NMR structure was reproduced from the PDB file 2L41 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref041" target="_blank">41</a>] using MacPyMOL software [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.ref062" target="_blank">62</a>] and altered and annotated using Adobe Photoshop and Illustrator CS4 (Adobe). (<b>B</b>) <i>nab3</i> RRM mutants, <i>nab3-R331A</i>, <i>nab3-F333A</i>, <i>nab3-S399A</i> do not suppress the <i>air1-C178R air2</i>Δ thermosensitive growth at 30°C. <i>nab3</i> RRM mutant, <i>nab3-S400A</i>, suppresses <i>air1-C178R air2</i>Δ thermosensitive growth at 30°C. The <i>air1-C178R air2</i>Δ cells containing vector, <i>NAB3</i> or <i>nab3 RRM</i> mutant <i>2μURA3</i> plasmid were spotted and grown at indicated temperatures. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s002" target="_blank">S2 Figs.</a> (<b>C</b>) All nab3 RRM mutant proteins, except nab3–11, are expressed in <i>air1-C178R air2</i>Δ cells to similar levels as Nab3. Lysates of <i>air1-C178R air2</i>Δ cells expressing Myc-tagged Nab3 or nab3 RRM mutants at 30°C were analyzed by immunoblotting to detect Myc-tagged proteins and 3-phosphoglycerate kinase (Pgk1) as a loading control. (<b>D</b>) The nab3-R331A and nab3-S399A RRM mutant proteins show binding to Nrd1 similar to wild-type Nab3. TAP-tagged Nrd1 was precipitated from lysates of <i>NRD1-TAP</i> cells expressing Myc-tagged Nab3, nab3-R331A or nab3-S399A and bound (B), unbound (U), and input fractions were analyzed by immunoblotting to detect Nab3-Myc proteins, Nrd1-TAP proteins and 3-phosphoglycerate kinase (Pgk1) as a loading control. The percentage of bound Nab3 relative to input protein and bound wild-type Nab3 (% Bound) is shown below the bound lanes. The percentage of input Nab3 protein relative to input wild-type Nab3 protein (% Input) is shown below the input lanes. The percentages of protein were calculated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#sec015" target="_blank">Materials and Methods</a>. Quantitation refers to specific experiment shown but is representative of multiple experiments. The original immunoblot is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005044#pgen.1005044.s003" target="_blank">S3A Fig.</a> (<b>E</b>) <i>nab3</i>Δcells expressing <i>nab3</i> RRM mutants, nab3-R331A, nab3-F333A or nab3-S399A, are not viable, but <i>nab3</i>Δ cells expressing <i>nab3-S400A</i> RRM mutant or <i>nab3-</i>Δ <i>NBD</i> mutant are viable. <i>nab3</i>Δ cells expressing <i>nab3–11</i> RRM mutant show a severe growth defect. <i>nab3</i>Δ cells containing <i>NAB3 URA3</i> maintenance plasmid and vector, <i>NAB3</i>, <i>nab3 RRM</i> mutant or <i>nab3-</i>Δ <i>NBD</i> mutant 2<i>μ HIS3</i> plasmid were spotted on control/5-FOA and grown at indicated temperatures.</p