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

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).