Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection

Author(s)

Lorenzon, Lucas ; Quilles Jr, Jose C. ; Campagnaro, Gustavo Daniel ; Orsine, Lissur Azevedo ; Almeida, Leticia ; Veras, Flavio ; Miserani Magalhaes, Rubens Daniel ; Diniz, Juliana Alcoforado ; Ferreira, Tiago Rodrigues ; Cruz, Angela Kaysel

• Document type: Journal article
• Source: ACS INFECTIOUS DISEASES; v. 8, n. 3, p. 17-pg., 2022-03-11.

Abstract

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L. braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions. (AU)

• FAPESP's process: 21/10043-5 - Comparative computational analyses of transcriptomes and large-scale identification of interactions between RNA-binding proteins and ncRNAs and repair/recombination proteins and DNA
• Grantee: Lissur Azevedo Orsine
• Support Opportunities: Scholarships in Brazil - Technical Training Program - Technical Training
• FAPESP's process: 20/00088-9 - Epigenetic regulation of Leishmania gene expression
• Grantee: José Carlos Quilles Junior
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 16/00969-0 - Studying the role of arginine methyltransferases in Leishmania braziliensis
• Grantee: Lucas Bigolin Lorenzon
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/14657-0 - Methylation mediated by PRMT7 on the differentiation process of promastigotes into amastigotes and the parasite Leishmania major virulence
• Grantee: Juliana Alcoforado Diniz
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/13618-8 - Regulating the trans-regulators: investigating the PRMT7 molecular pathway as an epigenetic regulator of Leishmania virulence
• Grantee: Angela Kaysel Cruz
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/14398-0 - UK:Brazil Joint Centre Partnership in Leishmaniasis (JCPiL)
• Grantee: Angela Kaysel Cruz
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 20/02372-6 - Actions coordinated by the enzyme Arginine Methyltransferase 5 of Leishmania braziliensis
• Grantee: Gustavo Daniel Campagnaro
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 19/18607-5 - Computational identification and structural characterization of non-coding RNA in Leishmania braziliensis
• Grantee: Rubens Daniel Miserani Magalhães
• Support Opportunities: Scholarships in Brazil - Post-Doctoral