Biology subjects

Wallace, E. W. J.

Publications and source records attributed to Wallace, E. W. J..

8 recordsLinked to original sources

bioRxiv2026

Endosomal hitchhiking and NDR kinase signaling coordinate SsdA-mRNP localization

Transport and regulation of messenger ribonucleoprotein complexes (mRNPs) is critical for spatial control of gene expression within cells. How mRNPs are trafficked in hyphae of different filamentous fungi remains poorly understood. Here we investigate the transport of SsdA, the Aspergillus nidulans ortholog of budding yeast RNA-binding protein Ssd1, which is thought to function in translational repression. SsdA forms cytoplasmic puncta that colocalize with the poly(A)-binding protein FabM, marking them as mRNPs, and punctum formation depends on conserved RNA-binding residues of SsdA. SsdA puncta move bidirectionally along microtubules by hitchhiking on early endosomes, and this requires the adaptor proteins PxdA and DipA--machinery previously associated exclusively with peroxisome transport. SsdA puncta are largely depleted from hyphal tip-proximal regions, despite an abundance of early endosomes near hyphal tips, and the length of SsdA puncta depletion zones correlates with hyphal growth rate. We show that acute inhibition of Nuclear Dbf2-related (NDR)-family protein kinase CotA causes rapid accumulation of SsdA puncta near hyphal tips, indicating that puncta depletion depends on CotA activity. Mutation of predicted CotA phosphorylation sites within SsdA to nonphosphorylatable residues also leads to accumulation of SsdA puncta near tips, while mutation to phosphomimetic residues disrupts puncta entirely. Together, our findings establish SsdA-containing mRNPs as a new endosomal hitchhiking cargo in A. nidulans and reveal that NDR kinase signaling spatially regulates SsdA-mRNP distribution during normal polarized growth.

cell biology↗

bioRxiv2025

Antifungal exposure can enhance Candida glabrata pathogenesis

Azole antifungal drugs directly inhibit lanosterol 14-[a]-demethylase and indirectly affect the expression of metabolic, transmembrane transporter, and cell wall organization genes in fungal pathogens. It is not known how these indirect azole effects depend on dose, timing, and specific azole used, or how they influence host interactions. Candida glabrata (recently renamed Nakaseomyces glabratus) is the second leading cause of candidiasis, and clinical strains have high rates of intrinsic resistance to azoles. We investigated the early responses of reference strains BG2 and CBS138 to sub-inhibitory doses of fluconazole and voriconazole, and particularly, how these responses affect host-pathogen interactions. Cell wall profiling and transcriptomic data revealed highly similar responses for each strain to both azoles, including the upregulation of several virulence factors, such as yapsins. We also observed significant increases in CBS138 survival in macrophages and increased virulence in Galleria mellonella after voriconazole exposure. Using a combination of pharmacological inhibition of calcium ion channels and deletion strains, we determined that voriconazole-enhanced virulence requires a yapsin protease, YPS1, and is regulated via the calcineurin pathway and the cell wall integrity pathway, both of which regulate YPS1 expression. We also observed that voriconazole treatment significantly reduced the virulence of the bck1{Delta} strain in G. mellonella, suggesting that inhibitors of the cell wall integrity pathway might potentiate azole activity by improving susceptibility to host killing. Our study provides new insight into short-term azole adaptation in C. glabrata, and importantly demonstrates that sub-inhibitory azole exposure can induce virulence factors and alter fungal pathogenesis. Article summaryAntifungal drugs indirectly affect essential fungal cell processes, but we lack an understanding of how drug-induced changes affect fungal pathogenesis. We investigated how Candida glabrata adapts when exposed to azole drugs in terms of cell wall and transcriptional changes. Reference strains had similar transcriptional changes in response to azoles, but azole-treated CBS138 survived better in immune cells and caused more host death than untreated cells, suggesting that short-term azole treatment can significantly affect pathogenesis. Voriconazole-enhanced disease requires the calcineurin and cell wall integrity pathways and the virulence factor, YPS1, but could be blocked by a calcium ion channel inhibitor. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/667834v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@157eaforg.highwire.dtl.DTLVardef@13050c0org.highwire.dtl.DTLVardef@10593d0org.highwire.dtl.DTLVardef@12b663e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Transcriptome-wide mRNA condensation precedes stress granule formation and excludes stress-induced transcripts

Stress-induced mRNP condensation is conserved across eukaryotes, resulting in stress granule formation under intense stresses, yet the mRNA composition and function of these condensates remain unclear. Exposure of ribosome-free mRNA following stress is thought to cause condensation and stress granule formation through mRNA-sequence-dependent interactions, leading to disproportionate condensation of long mRNAs. Here we show that, in striking contrast, virtually all mRNAs condense in response to multiple stresses in budding yeast with minor length dependence and often without stress granule formation. New transcripts escape mRNP condensation, enabling their selective translation. Inhibiting translation initiation causes formation of mRNP condensates that are distinct from stress granules and P-bodies; these translation-initiation-inhibited condensates (TIICs) are omnipresent, even in unstressed cells. Stress-induced mRNAs are excluded from TIICs due to the timing of their expression, indicating determinants of escape that are independent of sequence. Together, our results reveal a previously undetected level of translation-linked molecular organization and stress-responsive regulation.

cell biology↗

bioRxiv2023

choros: correction of sequence-based biases for accurate quantification of ribosome profiling data

Ribosome profiling quantifies translation genome-wide by sequencing ribosome-protected fragments, or footprints. Its single-codon resolution allows identification of translation regulation, such as ribosome stalls or pauses, on individual genes. However, enzyme preferences during library preparation lead to pervasive sequence artifacts that obscure translation dynamics. Widespread over- and under-representation of ribosome footprints can dominate local footprint densities and skew estimates of elongation rates by up to five fold. To address these biases and uncover true patterns of translation, we present choros, a computational method that models ribosome footprint distributions to provide bias-corrected footprint counts. choros uses negative binomial regression to accurately estimate two sets of parameters: (i) biological contributions from codon-specific translation elongation rates; and (ii) technical contributions from nuclease digestion and ligation efficiencies. We use these parameter estimates to generate bias correction factors that eliminate sequence artifacts. Applying choros to multiple ribosome profiling datasets, we are able to accurately quantify and attenuate ligation biases to provide more faithful measurements of ribosome distribution. We show that a pattern interpreted as pervasive ribosome pausing near the beginning of coding regions is likely to arise from technical biases. Incorporating choros into standard analysis pipelines will improve biological discovery from measurements of translation.

bioinformatics↗

bioRxiv2021

Probing 3'UTRs as modular regulators of gene expression

Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.

systems biology↗

bioRxiv2020

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

The conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 [A] resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

biochemistry↗

bioRxiv2020

Repeated evolution of inactive pseudonucleases in a fungal branch of the Dis3/RNase II family of nucleases

The RNase II family of 3'-5' exoribonucleases are present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like "pseudonucleases". These function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. Here, we show how these pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. The N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the non-nuclease function require this region. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid "titan" growth stages and yet retains an active site sequence signature. We propose that that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, while the RNase activity was lost repeatedly in independent lineages.

molecular biology↗

bioRxiv2019

Start codon context controls translation initiation in the fungal kingdom

Eukaryotic protein synthesis initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but studies of S. cerevisiae suggest this context is of little importance in fungi. We tested this concept in two pathogenic Cryptococcus species by genome-wide mapping of translation and of mRNA 5 and 3 ends. We observed that upstream open reading frames (uORFs) are a major contributor to translation repression, that uORF use depends on the Kozak sequence context of its start codon, and that uORFs with strong contexts promote nonsense-mediated mRNA decay. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Further analysis shows that such dual-localization is also predicted to be common in Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that programs the expression and structures of proteins in fungi.

genomics↗