Approaches for studying mRNA decay mediated by SIDER2 retroposons in Leishmania

Regulated mRNA turnover is a highly important process in the control of gene expression in Leishmania and related trypanosomatid protozoa, as these organisms lack control at the level of transcription initiation. A large number of Leishmania transcripts harbor in their 3'UTRs two phylogenetically distinct subfamilies of extinct Short Interspersed DEgenerate Retroposons (SIDER1 and SIDER2) that are involved in posttranscriptional regulation of gene expression. We have shown recently that members of the SIDER2 subfamily promote mRNA destabilization and that degradation of SIDER2-containing mRNAs is initiated by site-specific endonucleolytic cleavage within the second 79-nt SIDER2 signature sequence without prior shortening of the poly(A) tail. Here, we describe experimental procedures for studying the mechanism of SIDER2-mediated mRNA decay. These include RNase protection assays to identify in vivo-generated mRNA decay intermediates following endonucleolytic cleavage, primer extension analysis to precisely map the site(s) of cleavage within SIDER2, and deadenylation assays to assess the polyadenylation state of unstable SIDER2-containing mRNAs in Leishmania.