Efecto de la sobreexpresión del factor de transcripción EhMyb10 y el factor de splicing EhCDC5-like sobre el transcriptoma de Entamoeba histolytica

Abstract

RESUMEN:

El estudio investiga cómo Entamoeba histolytica regula su transcriptoma ante diversas condiciones, como virulencia y estrés, centrándose en las proteínas de unión a DNA MYB, específicamente EhMyb10 y EhCDC5. Se identificaron 147 genes potencialmente regulados cotranscripcionalmente mediante análisis in silico y se evaluó su sobreexpresión en trofozoítos, mostrando colocalización en el núcleo. Se encontraron 61 genes compartidos que modificaron su expresión tras la sobreexpresión de las proteínas, relacionados con metabolismo de RNA y virulencia. Además, la sobreexpresión afectó el splicing, sugiriendo un efecto sinérgico entre EhMyb10 y EhCDC5.

ABSTRACT:

“Entamoeba histolytica, regulates and modifies its transcriptome in response to different events such as virulence, encystment, and stress responses. This dynamic suggests that splicing and transcription processes may occur concurrently; this is referred to as cotranscriptional regulation. The MYB DNA binding domain proteins (DBD-MYB) belong to the most abundant families related to transcriptional regulation. EhMyb10 and EhCDC5 were selected from this family to determine their role in cotranscriptional regulation in E. histolytica, evaluating their functions as transcription and splicing factors, respectively. Initially, we performed an in silico analysis of the parasite's 8,201 genes, finding 147 genes with introns and the Myb recognition element (MRE) in their promoter region, which suggests that these genes may be targets of these proteins. In addition, we transiently overexpressed these proteins individually and in combination in E. histolytica trophozoites. Overexpression was evaluated through RT-qPCR, WB, and immunofluorescence assays. Through these assays, we determined that EhMyb10 and EhCDC5 are localized in the nucleus and cytoplasm and that they colocalize in the nucleus. EhCDC5 was detected with a molecular weight of ~ 30 kDa and EhMyb10 with ~40 kDa, which suggests the presence of postranslational modifications that could be necessary for their regulation. Subsequently, we examined the splicing patterns of four distinct genes that underwent modification in various overexpression conditions. Finally, through an RNAseq analysis, it was observed that 179, 243, and 115 genes modified their expression in EhMyb10ox, EhCDC5ox, and in combined conditions, respectively. From these genes, 61 were shared in all conditions, which could be target genes of cotranscriptional regulation. These genes are related to different functions, such as RNA metabolism, transcriptional regulation, and virulence properties”.