Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA.
The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.
Zwicky
Roman
R
Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Müntener
Kathrin
K
Csucs
Gabor
G
Goldring
Mary B
MB
Baici
Antonio
A
D020413:3' Untranslated Regions / Q000032:analysis / Q000235:genetics; D020121:5' Untranslated Regions / Q000032:analysis / Q000235:genetics; D017398:Alternative Splicing; D002401:Cathepsin B / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D019902:Chondrocytes / Q000378:metabolism; D017931:DNA Primers; D015536:Down-Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D002401:Cathepsin B