Characterization of the 5′ region of the Leishmania infantum LORIEN/MAT2 gene cluster and role of LORIEN flanking regions in post-transcriptional regulation

LORIEN (encoding a protein that contains a SP-RING/Miz zinc-finger motif present in a group of proteins involved in the Small Ubiquitin-related Modifier -SUMO- conjugation pathway) and MAT2 (encoding the methionine adenosyltransferase -MAT-) genes are arranged as two alternating copies in a head-to-tail configuration, with the LORIEN gene as the first copy of the cluster. The 5880 bp preceding the first LORIEN gene copy were compared to the same region of L. major, showing a 93% identity between them. Bioinformatic analysis of this region predicted the presence of a 747-bp ORF encoding a hypothetical protein of 248 amino acids. Transcription of this ORF was confirmed by run-on assays and RT-PCR. Expression of the LORIEN gene was tested in both the promastigote and amastigote stages. Transcription arrest evidenced that LORIEN mRNA stability was very similar in both stages of the parasite life cycle. Protein synthesis inhibition by cycloheximide led to an increase in the steady-state levels of LORIEN transcripts only during the promastigote stage, pointing out to the existence of different stage-dependent mechanisms operating on the post-transcriptional regulation of this gene. The role of the LORIEN untranslated regions (5′UTR and 3′UTR) in post-transcriptional regulation was analysed using the luciferase (luc) reporter gene. Results evidenced that the 5′UTR was responsible for a low reporter gene expression, whereas the intergenic region (IR) between LORIEN and MAT2 genes provided high luc levels. However, the 3′UTR seemed to lack regulatory elements. Basing on these results, a model of regulation for the LORIEN gene is proposed.