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Protein interactions and phosphorylation status of Maf1 during optimal growth and stress conditions in the human parasite Leishmania major

ABSTRACT: Maf1 was originally described as a repressor of RNA polymerase III transcription in response to stress conditions in yeast. Recent work has shown that Maf1 is involved in several functions across eukaryotes. Here we characterize Maf1 in Leishmania major (LmMaf1), a protozoan parasite that exhibits unconventional mechanisms of gene expression. Despite sequence divergence, LmMaf1 contains the conserved domains A, B and C. Four phosphorylated residues were detected in domains B and C in mid-logarithmic phase parasites by mass spectrometry. This is an unexpected result, since phosphorylation has not been reported in these domains in other organisms. Notably, no phosphorylated amino acids were found in stationary phase parasites, suggesting that nutrient stress might induce dephosphorylation of LmMaf1. Indirect immunofluorescence assays with a cell line that expresses LmMaf1 fused to a PTP tag showed that Maf1 mainly localizes to the nucleus. Interestingly, unlike other species, the LmMaf1 subcellular localization did not change in heat-shocked and stationary-phase parasites. Tandem affinity purifications were executed to identify the proteins that interact with LmMaf1 under optimal growth and heat stress. Although similar groups of proteins were identified in both normal and heat-stressed cells, the relative abundance of several of them showed important changes. For example, most RNA polymerase subunits increased significantly their relative abundance after heat shock. Altogether, these results strongly suggest the involvement of Maf1 in diverse roles that help to maintain general cellular homeostasis both under normal and stressful conditions in L. major, including transcription control, post-transcriptional regulation of mRNA expression, lipogenesis, and ribosomal biogenesis.

INSTRUMENT(S):

ORGANISM(S): Leishmania Major Strain Friedlin

SUBMITTER: Ebbing de Jong

LAB HEAD: Santiago Calvillo-Martinez

PROVIDER: PXD067545 | Pride | 2025-11-24

REPOSITORIES: Pride

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			Action	DRS
	20211123_SC_LMmaf1-1_high.mzML	Mzml
	20211123_SC_LMmaf1-1_high.mzid	Mzid
	20211123_SC_LMmaf1-1_high.raw	Raw
	20211123_SC_LMmaf1-2_high.mzML	Mzml
	20211123_SC_LMmaf1-2_high.mzid	Mzid

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Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions. Transcriptomes of two yeast species under mid-log phase, early stationary phase, and after heat shock treatment were generated by Illumina HiSeq 2000 paired-end sequencing

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Protein interactions and phosphorylation status of Maf1 during optimal growth and stress conditions in the human parasite Leishmania major

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