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

Optimizing HIV testing services in sub-Saharan Africa: cost and performance of verification testing with HIV self-tests and tests for triage.

INTRODUCTION: Strategies employing a single rapid diagnostic test (RDT) such as HIV self-testing (HIVST) or "test for triage" (T4T) are proposed to increase HIV testing programme impact. Current guidelines recommend serial testing with two or three RDTs for HIV diagnosis, followed by retesting with the same algorithm to verify HIV-positive status before anti-retroviral therapy (ART) initiation. We investigated whether clients presenting to HIV testing services (HTS) following a single reactive RDT must undergo the diagnostic algorithm twice to diagnose and verify HIV-positive status, or whether a diagnosis with the setting-specific algorithm is adequate for ART initiation. METHODS: We calculated (1) expected number of false-positive (FP) misclassifications per 10,000 HIV negative persons tested, (2) positive predictive value (PPV) of the overall HIV testing strategy compared to the WHO recommended PPV ≥99%, and (3) expected cost per FP misclassified person identified by additional verification testing in a typical low-/middle-income setting, compared to the expected lifetime ART cost of $3000. Scenarios considered were as follows: 10$5879, $3770, and$24,259 respectively. Results were sensitive to assumptions about accuracy of self-reported reactive results and whether reactive triage test results influenced biased interpretation of subsequent RDT results by the HTS provider. CONCLUSIONS: Diagnosis with the full algorithm following presentation with a reactive triage test is expected to achieve PPV above the 99% threshold. Continuing verification testing prior to ART initiation remains recommended, but HIV testing strategies involving HIVST and T4T may provide opportunities to maintain quality while increasing efficiency as part of broader restructuring of HIV testing service delivery

Putting It Together: AIDS and the Millennium Development Goals

Failure to halt and reverse the HIV/AIDS epidemic, say the authors, will continue to jeopardize progress on achieving a wide range of the MDGs

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

An Endoribonuclease Functionally Linked to Perinuclear mRNP Quality Control Associates with the Nuclear Pore Complexes

Nuclear mRNA export is a crucial step in eukaryotic gene expression, which is in yeast coupled to cotranscriptional messenger ribonucleoprotein particle (mRNP) assembly and surveillance. Several surveillance systems that monitor nuclear mRNP biogenesis and export have been described, but the mechanism by which the improper mRNPs are recognized and eliminated remains poorly understood. Here we report that the conserved PIN domain protein Swt1 is an RNA endonuclease that participates in quality control of nuclear mRNPs and can associate with the nuclear pore complex (NPC). Swt1 showed endoribonuclease activity in vitro that was inhibited by a point mutation in the predicted catalytic site. Swt1 lacked clear sequence specificity but showed a strong preference for single-stranded regions. Genetic interactions were found between Swt1 and the THO/TREX and TREX-2 complexes, and with components of the perinuclear mRNP surveillance system, Mlp1, Nup60, and Esc1. Inhibition of the nuclease activity of Swt1 increased the levels and cytoplasmic leakage of unspliced aberrant pre-mRNA, and induced robust nuclear poly(A)+ RNA accumulation in mlp1Δ and esc1Δ strains. Overexpression of Swt1 also caused strong nuclear poly(A)+ RNA accumulation. Swt1 is normally distributed throughout the nucleus and cytoplasm but becomes concentrated at nuclear pore complexes (NPCs) in the nup133Δ mutant, which causes NPC clustering and defects in mRNP export. The data suggest that Swt1 endoribonuclease might be transiently recruited to NPCs to initiate the degradation of defective pre-mRNPs or mRNPs trapped at nuclear periphery in order to avoid their cytoplasmic export and translation