Functional analysis of cis-acting DNA elements required for expression of the SL RNA gene in the parasitic protozoan Leishmania amazonensis.

Abstract

DNA sequences, that control expression of the spliced leader (SL) RNA gene in the parasitic protozoan Leishmania amazonensis, were mapped by block substitution mutagenesis. In the absence of a functional in vitro system for transcription, no promoter elements have yet been identified in this organism. We therefore developed an alternative in vivo approach, in which the SL RNA gene was tagged and then subjected to a series of linker scanning mutations. Each tagged and mutated SL RNA construct was introduced into parasite cells via the pX transfection vector, and was examined for expression of the tagged SL RNA followed by characterization of its transcriptional start site. The replacement of a critical DNA element was expected to prevent expression of the tagged SL RNA. We found that the putative SL RNA promoter is complex and includes two elements: one is located upstream to the coding region, between positions -30 to -70; and the other is located between -10 to +10, and includes transcribed sequences. In addition to the functional relationship between the SL RNA and vertebrate U snRNAs, we found structural similarities in their regulatory elements, which may possibly indicate a common evolutionary ancestry for these molecules.