Post-translational modification directs nuclear and hyphal tip localization of Candida albicans mRNA-binding protein Slr1.

The morphological transition of the opportunistic fungal pathogen Candida albicans from budding to hyphal growth has been implicated in its ability to cause disease in animal models. Absence of SR-like RNA-binding protein Slr1 slows hyphal formation and decreases virulence in a systemic candidiasis model, suggesting a role for post-transcriptional regulation in these processes. SR (serine-arginine)-rich proteins influence multiple steps in mRNA metabolism and their localization and function are frequently controlled by modification. We now demonstrate that Slr1 binds to polyadenylated RNA and that its intracellular localization is modulated by phosphorylation and methylation. Wildtype Slr1-GFP is predominantly nuclear, but also co-fractionates with translating ribosomes. The non-phosphorylatable slr1-6SA-GFP protein, in which six serines in SR/RS clusters are substituted with alanines, primarily localizes to the cytoplasm in budding cells. Intriguingly, hyphal cells display a slr1-6SA-GFP focus at the tip near the Spitzenkörper, a vesicular structure involved in molecular trafficking to the tip. The presence of slr1-6SA-GFP hyphal tip foci is reduced in the absence of the mRNA-transport protein She3, suggesting that unphosphorylated Slr1 associates with mRNA-protein complexes transported to the tip. The impact of SLR1 deletion on hyphal formation and function thus may be partially due to a role in hyphal mRNA transport.

Messenger RNA transport in the opportunistic fungal pathogen Candida albicans.

Candida albicans, a common commensal fungus, can cause disease in immunocompromised hosts ranging from mild mucosal infections to severe bloodstream infections with high mortality rates. The ability of C. albicans cells to switch between a budding yeast form and an elongated hyphal form is linked to pathogenicity in animal models. Hyphal-specific proteins such as cell-surface adhesins and secreted hydrolases facilitate tissue invasion and host cell damage, but the specific mechanisms leading to asymmetric protein localization in hyphae remain poorly understood. In many eukaryotes, directional cytoplasmic transport of messenger RNAs that encode asymmetrically localized proteins allows efficient local translation at the site of protein function. Over the past two decades, detailed mechanisms for polarized mRNA transport have been elucidated in the budding yeast Saccharomyces cerevisiae and the filamentous fungus Ustilago maydis. This review highlights recent studies of RNA-binding proteins in C. albicans that have revealed intriguing similarities to and differences from known fungal mRNA transport systems. I also discuss outstanding questions that will need to be answered to reach an in-depth understanding of C. albicans mRNA transport mechanisms and the roles of asymmetric mRNA localization in polarized growth, hyphal function, and virulence of this opportunistic pathogen.

SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

Identification of methylated proteins in the yeast small ribosomal subunit: a role for SPOUT methyltransferases in protein arginine methylation.

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation of Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.

Specific sequences within arginine-glycine-rich domains affect mRNA-binding protein function.

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine-glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.

Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling.

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Food porn.

Since the term first appeared, food porn has typically referred to watching others cook on television or gazing at unattainable dishes in glossy magazines without actually cooking oneself. This forum seeks to revisit this notion of food porn that is mostly taken for granted in both popular and scholarly literature. It offers a brief perspective of the appearance and use of the term food porn to examine how it came to be a term used mostly by commentators rather than by people actively engaged in the world of cooking. Practitioners (chefs and a food television producer) and academics address whether or not food porn exists, what shape it might take, what purpose it might serve, and/or what usefulness it might have, showing that these contentious issues are more complex than the ease with which the term is used might let on.

Protein arginine methylation in Candida albicans: role in nuclear transport.

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and omega-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Delta strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Delta/rmt2Delta strain, as well as biochemical evidence for additional cryptic PRMTs.

3 Diverse roles of protein arginine methyltransferases.

Methylation of arginine residues within proteins results in a more subtle change than phosphorylation, but one that also has profound impacts on eukaryotic organisms. The rapidly expanding list of protein arginine methyltransferases (PRMTs) and PRMT substrates has implicated arginine methylation in a wide variety of cellular processes. This chapter explores the diverse functions of PRMTs by examining evidence for effects of arginine methylation and PRMTs on specific cellular processes, and connecting the data to molecular mechanisms that have been proposed to explain these effects. First, effects of arginine methylation on intermolecular interactions are addressed, focusing on how methylation affects protein-nucleic acid and protein-protein interactions. Next, the numerous linksamong PRMTs, arginine methylation, and cellular processes are described, including roles of PRMTs and methylation in transcription, cell signaling, protein transport, pre-mRNA splicing, ribosome assembly, and DNA damage repair. Finally, the broader implications of arginine methylation are considered by examining the connections between PRMTs and their substrates and development, differentiation, and disease.

Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions.

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels of methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmt1. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmt1, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins.

Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors.

Hmt1 is the major type I arginine methyltransferase in the yeast Saccharomyces cerevisiae and facilitates the nucleocytoplasmic transport of mRNA-binding proteins through their methylation. Here we demonstrate that Hmt1 is recruited during the beginning of the transcriptional elongation process. Hmt1 methylates Yra1 and Hrp1, two mRNA-binding proteins important for mRNA processing and export. Moreover, loss of Hmt1 affects interactions between mRNA-binding proteins and Tho2, a component of the TREX (transcription/export) complex that is important for transcriptional elongation and recruitment of mRNA export factors. Furthermore, RNA in situ hybridization analysis demonstrates that loss of Hmt1 results in slowed release of HSP104 mRNA from the sites of transcription. Genome-wide location analysis shows that Hmt1 is bound to specific functional gene classes, many of which are also bound by Tho2 and other mRNA-processing factors. These data suggest a model whereby Hmt1 affects transcriptional elongation and, as a result, influences recruitment of RNA-processing factors.