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

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

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

Functional overlap between conserved and diverged KH domains in Saccharomyces cerevisiae SCP160

The K homology (KH) domain is a remarkably versatile and highly conserved RNA-binding motif. Classical KH domains include a characteristic pattern of hydrophobic residues, a Gly-X-X-Gly (GXXG) segment, and a variable loop. KH domains typically occur in clusters, with some retaining their GXXG sequence (conserved), while others do not (diverged). As a first step towards addressing whether GXXG is essential for KH-domain function, we explored the roles of conserved and diverged KH domains in Scp160p, a multiple-KH-domain-containing protein in Saccharomyces cerevisiae. We specifically wanted to know (1) whether diverged KH domains were essential for Scp160p function, and (2) whether diverged KH domains could functionally replace conserved KH domains. To address these questions, we deleted and/or interchanged conserved and diverged KH domains of Scp160p and expressed the mutated alleles in yeast. Our results demonstrated that the answer to each question was yes. Both conserved and diverged KH domains are essential for Scp160p function, and diverged KH domains can function in place of conserved KH domains. These findings challenge the prevailing notions about the requisite features of a KH domain and raise the possibility that there may be more functional KH domains in the proteome than previously appreciated

The Gly2019Ser mutation in LRRK2 is not fully penetrant in familial Parkinson's disease: the GenePD study

<p>Abstract</p> <p>Background</p> <p>We report age-dependent penetrance estimates for leucine-rich repeat kinase 2 (<it>LRRK2</it>)-related Parkinson's disease (PD) in a large sample of familial PD. The most frequently seen <it>LRRK2 </it>mutation, Gly2019Ser (G2019S), is associated with approximately 5 to 6% of familial PD cases and 1 to 2% of idiopathic cases, making it the most common known genetic cause of PD. Studies of the penetrance of <it>LRRK2 </it>mutations have produced a wide range of estimates, possibly due to differences in study design and recruitment, including in particular differences between samples of familial PD versus sporadic PD.</p> <p>Methods</p> <p>A sample, including 903 affected and 58 unaffected members from 509 families ascertained for having two or more PD-affected members, 126 randomly ascertained PD patients and 197 controls, was screened for five different <it>LRRK2 </it>mutations. Penetrance was estimated in families of <it>LRRK2 </it>carriers with consideration of the inherent bias towards increased penetrance in a familial sample.</p> <p>Results</p> <p>Thirty-one out of 509 families with multiple cases of PD (6.1%) were found to have 58 <it>LRRK2 </it>mutation carriers (6.4%). Twenty-nine of the 31 families had G2019S mutations while two had R1441C mutations. No mutations were identified among controls or unaffected relatives of PD cases. Nine PD-affected relatives of G2019S carriers did not carry the <it>LRRK2 </it>mutation themselves. At the maximum observed age range of 90 to 94 years, the unbiased estimated penetrance was 67% for G2019S families, compared with a baseline PD risk of 17% seen in the non-<it>LRRK2</it>-related PD families.</p> <p>Conclusion</p> <p>Lifetime penetrance of <it>LRRK2 </it>estimated in the unascertained relatives of multiplex PD families is greater than that reported in studies of sporadically ascertained <it>LRRK2 </it>cases, suggesting that inherited susceptibility factors may modify the penetrance of <it>LRRK2 </it>mutations. In addition, the presence of nine PD phenocopies in the <it>LRRK2 </it>families suggests that these susceptibility factors may also increase the risk of non-<it>LRRK2</it>-related PD. No differences in penetrance were found between men and women, suggesting that the factors that influence penetrance for <it>LRRK2 </it>carriers are independent of the factors which increase PD prevalence in men.</p

Ontogeny of Toll-Like and NOD-Like Receptor-Mediated Innate Immune Responses in Papua New Guinean Infants

Studies addressing the ontogeny of the innate immune system in early life have reported mainly on Toll-like receptor (TLR) responses in infants living in high-income countries, with little or even no information on other pattern recognition receptors or on early life innate immune responses in children living under very different environmental conditions in less-developed parts of the world. In this study, we describe whole blood innate immune responses to both Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor agonists including the widely used vaccine adjuvant ‘alum’ in a group of Papua New Guinean infants aged 1–3 (n = 18), 4–6 (n = 18), 7–12 (n = 21) and 13–18 (n = 10) months old. Depending on the ligands and cytokines studied, different age-related patterns were found: alum-induced IL-1β and CXCL8 responses were found to significantly decline with increasing age; inflammatory (IL-6, IL-1β, IFN-γ) responses to TLR2 and TLR3 agonists increased; and IL-10 responses remained constant or increased during infancy, while TNF-α responses either declined or remained the same. We report for the first time that whole blood innate immune responses to the vaccine adjuvant alum decrease with age in infancy; a finding that may imply that the adjuvant effect of alum in pediatric vaccines could be age-related. Our findings further suggest that patterns of innate immune development may vary between geographically diverse populations, which in line with the ‘hygiene hypothesis’ particularly involves persistence of innate IL-10 responses in populations experiencing higher infectious pressure

Regulation of base excision repair: Ntg1 nuclear and mitochondrial dynamic localization in response to genotoxic stress

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage