The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Abstract

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5∆ mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5∆ mutant showed that Rmt5 is critical for the homeostasis of eIF2α and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5∆ clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5∆ phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA- binding proteins.

Arginine methylation of Puf4 drives diverse protein functions

“…By contrast, the inability of the methyl-deficient mutants to complement the thermotolerance defect of the puf4 Δ strain at 38°C suggests that the modification of arginines in Puf4 drives temperature-specific protein function. This is especially intriguing because RMT5 (which interacts with Puf4) is the only C. neoformans arginine methyltransferase for which deletion similarly eliminates thermotolerance at elevated temperatures 25 . Thus, we speculate that Rmt5 acts on Puf4 to establish thermotolerance.…”

“…RNA quality was determined by investigating integrity on an RNA gel and using a bioanalyzer prior to sequencing. RNA sequencing was performed as described previously 25 . RNA samples were submitted to Genewiz for library preparation and Illumina sequencing.…”

“…The ability to leverage dynamic and often reversible PTMs may impart agility for post-transcriptional gene regulation 23,24 . Our recent work to characterize RMTs in C. neoformans revealed that one of its four PUF proteins, Puf4, interacts with Rmt5, suggesting that its function is controlled through methylation 25 . Puf4 regulates mRNAs involved in cell wall biosynthesis and is involved in the splicing of HXL1, the major endoplasmic reticulum (ER) stress transcription factor 26,27 .…”

Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination

“…Following on-bead digestion, LC-MS was performed as described previously (Kalem et al, 2022). Within each experimental condition and for each independent experiment, the total ion chromatograph (TIC) intensity for each protein was calculated relative to the total TIC intensity for all the biotinylated proteins pulled down by streptavidin-affinity purification (AP), so that we compare the percentage contribution of each protein to the total biotinylated pool.…”