Unexplored Molecular Features of the Entamoeba histolytica RNA Lariat Debranching Enzyme Dbr1 Expression Profile

Jesús Valdés,Carlos Ortuño-Pineda,Odila Saucedo-Cárdenas,María S Mendoza-Figueroa

doi:10.3389/fcimb.2018.00228

Abstract

The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2′-5′ linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.

Highlights

The spliceosome mediates intron removal and exon ligation of pre-mRNA through two consecutive trans-esterification reactions, resulting in a 2′-5′ linked lariat and in mRNA molecules (Padgett et al, 1984; Ruskin et al, 1984; Ruskin and Green, 1985; Konarska et al, 2006; Smith et al, 2009)
These data and data from other organisms suggest that Dbr1 gene expression is finely tuned and conserved across eukaryotes
To determine the half-life of the Entamoeba histolytica Dbr1 (EhDbr1) mRNA, RNA polymerase II transcription was inhibited in E. histolityca trophozoites using Actinomycin D (Ayala et al, 1990; Lopez-Camarillo et al, 2003); after different incubation times with the drug, the total RNA was isolated, and the expression of EhDbr1 was analyzed by RT-Polymerase Chain Reactions (PCR)

Summary

Introduction

The spliceosome mediates intron removal and exon ligation of pre-mRNA through two consecutive trans-esterification reactions, resulting in a 2′-5′ linked lariat and in mRNA molecules (Padgett et al, 1984; Ruskin et al, 1984; Ruskin and Green, 1985; Konarska et al, 2006; Smith et al, 2009). Dbr protein sequences from different organisms show high homology (Nam et al, 1997; Kim et al, 2000; Kataoka et al, 2013), suggesting that Dbr both in structure and in function is phylogenetically conserved. Dbr enzymes belong to the metallophosphoesterase (MPE) family (Koonin, 1994), showing a conserved N-terminal domain, a C-terminal domain (CTD) that does not show sequence similarity to any other class of proteins; a third domain between them, the lariat recognition loop (LRL) adjacent to the active site, is not present in other MPEs (Kim et al, 2000). As MPEs, Dbr enzymes use divalent metals for their activity (Arenas and Hurwitz, 1987), such as manganese in the case of the Saccharomyces cerevisiae Dbr (ScDbr1) (Khalid et al, 2005) or Fe2+, Zn2+, or Mn2+ in the case of Entamoeba histolytica Dbr (EhDbr1) (Clark et al, 2016; Ransey et al, 2017), suggesting a different requirement of metal cofactors

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Conclusion