Regulation of genes encoding the major surface protease of Leishmania chagasi via mRNA stability

Abstract

The intercoding regions between many Leishmania sp. genes regulate their mRNA expression. The MSPL mRNA, encoding a subclass of the major surface protease (MSP) of Leishmania chagasi, increases in abundance, when protein synthesis is arrested, while α-tubulin (α-TUB) mRNA and most other mRNAs do not. We found that the intercoding region between MSPL-coding regions, when cloned downstream of the β-galactosidase reporter gene (β-GAL), caused β-GAL mRNA to increase 8- to 10-fold after inhibiting protein synthesis with cycloheximide. Stable L. chagasi transfectants containing hybrid MSPL/α-TUB intercoding regions cloned downstream of β-GAL were made. The α-TUB intercoding region induced high-level baseline β-GAL mRNA that increased only 1.3-fold after incubation with cycloheximide. In contrast, the MSPL intercoding region, as well as constructs containing nucleotides 303-505 from the MSPL 3′UTR, caused steady-state β-GAL mRNA levels in the absence of cycloheximide that were approximately 10% of α-TUB constructs. These levels increased between 4.4- and 13.2-fold after cycloheximide was added. Constructs containing half of this region (303-394 or 395-505) produced intermediate levels of β-GAL mRNA and intermediate levels of cycloheximide induction. The kinetics of cycloheximide induction of β-GAL mRNA was similar with region 303-505 constructs as with constructs bearing the entire endogenous MSPL intercoding region. Furthermore, region 303-505 increased reporter mRNA abundance after cycloheximide by increasing mRNA half-life. Hence, we have identified a 202-nucleotide region within the MSPL 3′UTR that is in part responsible for cycloheximide induction. We hypothesize that this region may interact with labile regulatory protein factor(s). © 2005 Elsevier B.V. All rights reserved.