Transcriptome and proteome changes triggered by overexpression of the transcriptional regulator Maf1 in the human pathogen Leishmania major

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 42% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Maf1最初被描述为酵母中RNA聚合酶III（RNAP III）转录的阻遏因子，在真核生物中参与多种生物学功能。然而，目前关于原生动物寄生虫中Maf1的研究资料仍较为匮乏。为启动对利什曼原虫（Leishmania major）中Maf1的研究，我们构建了一株过表达该蛋白的细胞系。Maf1过表达会导致转运RNA（tRNA）、5S核糖体RNA（5S rRNA）以及U4小核RNA（U4 snRNA）的丰度显著降低，证明Maf1可调控利什曼原虫中的RNAP III活性。为进一步探究Maf1在该微生物中的功能，我们通过RNA测序（RNA-sequencing）和无标记定量质谱（label-free quantitative mass spectrometry）分析了Maf1过表达引发的全局转录组与蛋白质组变化。与野生型细胞相比，过表达细胞中共检测到1082个差异表达转录本（615个下调、467个上调）以及205个差异表达蛋白质（132个下调、73个上调）。转录组与蛋白质组结果的相关性为42%。基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析显示，差异表达的基因和蛋白质主要参与转录、细胞周期调控、脂质代谢与转运、核糖体生物发生、碳水化合物代谢以及细胞骨架修饰等过程。因此，我们的研究结果表明，正如其他物种中所报道的那样，Maf1参与调控利什曼原虫的上述所有生物学过程，这提示Maf1的功能在真核生物进化早期便已确立。值得注意的是，我们的数据还表明利什曼原虫Maf1参与了信使RNA（mRNA）的转录后调控，据我们所知，这一功能在其他生物体中尚未被报道过。