The 3′-untranslated region of the Ste20-like kinase SLK regulates SLK expression

Abstract

Ste20-like kinase, SLK, a germinal center kinase found in kidney epithelial cells, signals to promote apoptosis. Expression of SLK mRNA and protein and kinase activity are increased during kidney development and recovery from ischemic acute renal failure. The 3'-untranslated region (3'-UTR) of SLK mRNA contains multiple adenine and uridine-rich elements, suggesting that 3'-UTR may regulate mRNA stability. This was confirmed in COS cell transient transfection studies, which showed that expression of the SLK open-reading frame plus 3'-UTR mRNA was reduced by 35% relative to the open-reading frame alone. To further characterize the SLK-3'-UTR, this nucleotide sequence was subcloned downstream of enhanced green fluorescent protein (EGFP) cDNA. In COS, 293T, and glomerular epithelial cells, expression of EGFP mRNA and protein was markedly reduced in the presence of the SLK-3'-UTR. After transfection and subsequent addition of actinomycin D, EGFP mRNA remained stable in cells for at least 6 h, whereas EGFP-SLK-3'-UTR mRNA decayed with a half-life of approximately 4 h. A region containing five AUUUA motifs within the SLK-3'-UTR destabilized EGFP mRNA. Deletion of this region from the SLK-3'-UTR, in part, restored mRNA stability. By UV cross-linking and SDS-PAGE, the SLK-3'-UTR bound to protein(s) of approximately 30 kDa in extracts of COS cells, glomerular epithelial cells, and kidney. Cotransfection of HuR (a RNA binding protein of approximately 30 kDa) increased the steady-state mRNA level of EGFP-SLK-3'-UTR but not EGFP. Thus the SLK-3'-UTR may interact with kidney RNA-binding proteins to regulate expression of SLK mRNA during kidney development and after ischemic injury.