UPF1, a Conserved Nonsense-Mediated mRNA Decay Factor, Regulates Cyst Wall Protein Transcripts in Giardia lamblia

The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp) genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD) system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia

Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification

Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called “nonsense-mediated decay”, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 3′-untranslated region length alone

Farnesol Biosynthesis in Candida albicans: Cellular Response to Sterol Inhibition by Zaragozic Acid B

The dimorphic fungus Candida albicans produces farnesol as a quorum-sensing molecule that regulates cellular morphology. The biosynthetic origin of farnesol has been resolved by treating these cells with zaragozic acid B, a potent inhibitor of squalene synthase in the sterol biosynthetic pathway. Treatment with zaragozic acid B leads to an eightfold increase in the amount of farnesol produced by C. albicans. Furthermore, C. albicans cell extracts contain enzymatic activity to convert [(3)H]farnesyl pyrophosphate to [(3)H]farnesol. Many common antifungal antibiotics (e.g., zaragozic acids, azoles, and allylamines) target steps in sterol biosynthesis. We suggest that the fungicidal activity of zaragozic acid derives in large part from the accumulation of farnesol that accompanies the inhibition of sterol biosynthesis

Cytoplasmic mRNA surveillance pathways

International audienceDuring mRNA synthesis and maturation, the introduction of errors can strongly influence the expression of certain genes and/or the activity of the proteins for which they encode. To minimise these defects, eukaryotic cells have evolved several cytoplasmic and translation-dependent quality control pathways aimed at detecting and degrading mRNAs that would lead to the production of aberrant proteins. The nonsense-mediated mRNA decay pathway (NMD) clears cells from mRNAs harbouring premature in-frame stop codons. Two other pathways (NSD for nonstop decay and NGD for No-Go decay) degrade mRNAs on which ribosomes have stalled during elongation. In this chapter, we describe the current knowledge on the biological roles and molecular mechanisms of these surveillance pathways, which were mainly unravelled using baker’s yeast as model system