Date Published: July 13, 2017
Publisher: Public Library of Science
Author(s): Hiva Azizi, Tatiany P. Romão, Karen Santos Charret, Prasad K. Padmanabhan, Osvaldo P. de Melo Neto, Michaela Müller-McNicoll, Barbara Papadopoulou, Juan Mata.
We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3’UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.
Leishmania species cause a broad spectrum of vector-borne parasitic diseases, ranging from self-healing cutaneous to fatal visceral forms that are collectively termed as leishmaniasis. The disease has expanded to over 98 countries with more than 350 million people at risk and 2 million individuals infected annually [1, 2]. Leishmania is an early-branching unicellular eukaryote  that belongs to the Trypanosomatidae family. These organisms lack defined RNA pol II promoters and the typical general transcription factors and have most likely lost the ability to regulate gene expression at the level of transcription initiation. Instead, RNA polymerase II transcribes long polycistronic units that are further processed into individual mature mRNAs by two coupled RNA-processing reactions, namely, trans-splicing which adds the 39-nt spliced leader RNA to the 5′ terminus of all protein-encoding RNAs and 3′-polyadenylation [4, 5]. Thus, regulation of gene expression in these organisms occurs exclusively at the post-transcriptional level [6, 7].
We have reported previously a novel mechanism of regulated mRNA decay in Leishmania involving Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, located largely within 3’UTRs of Leishmania transcripts. We have shown that the second tandem 79-nt hallmark signature sequence (79-nt SII), conserved among all SIDER2 retroposons, is essential for regulation and that it mediates rapid mRNA turnover through endonucleolytic cleavage [13, 14]. In this study, we provide new insights into the sequence and structural requirements for efficient mRNA cleavage and degradation using mutational and in silico RNA structural studies to further delineate the cis-acting region within 79-nt SII.