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Identification and characterization of a novel human cathepsin L splice variant
Shivani Arora1, Shyam S Chauhan
1Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
Insights
A new human cathepsin L (hCATL) splice variant, hCATL AIII, has been identified. This variant is the most abundant and efficiently translated form in human cell lines, suggesting a role in cancer progression.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Human cathepsin L (hCATL) is involved in various physiological and pathological processes.
- hCATL is encoded by multiple mRNA species (A, AI, AII, B) with distinct 5' untranslated regions (UTRs).
- Previous variants (AI, AII) arise from alternative splicing of hCATL A, differing in exon 1 length.
Purpose of the Study:
- To identify and characterize novel splice variants of human cathepsin L.
- To investigate the abundance and translational efficiency of different hCATL splice variants.
- To explore the functional implications of a newly discovered splice variant in human cell lines and cancer.
Main Methods:
- Identification of a novel hCATL splice variant (hCATL AIII) through sequence analysis.
- In vitro transcription and coupled translation assays to assess protein production efficiency.
- Enzymatic activity measurements of translated products.
- Luciferase reporter gene assays to evaluate the regulatory role of the 5' UTR.
Main Results:
- A new splice variant, hCATL AIII, was identified, lacking 145 bases from the 3' end of exon 1 of hCATL A.
- hCATL AIII was the most abundant splice variant across five human cell lines.
- hCATL AIII demonstrated significantly higher translation efficiency (1.6-4.4 fold) compared to hCATL A, AI, and AII.
- Luciferase assays confirmed a 3.75-fold higher expression driven by the hCATL AIII UTR.
Conclusions:
- A novel, highly abundant, and efficiently translated hCATL splice variant (hCATL AIII) has been discovered.
- The UTR of hCATL AIII contains regulatory elements conferring translational advantage.
- The prevalence of hCATL AIII in malignant cells suggests its critical role in hCATL overexpression in cancer.
Abstract:
Human cathepsin L (hCATL) has been implicated in a variety of physiological and pathological processes. It was hitherto known to be encoded by four mRNA species, namely hCATL A, AI, AII and hCATL B, differing in their 5' untranslated regions (UTRs). Of these, hCATL A, AI and AII are produced by the alternative splicing of the same primary transcript. HCATL AI and hCATL AII, lack 27 and 90 bases, respectively, from the 3' end of exon 1 of hCATL A. The present study describes the identification of a new splice variant hCATL AIII, which similarly lacks 145 bases from the 3' end of exon 1 of hCATL A. It is produced by the splicing out of 136-280 bases of the first exon in addition to intron 1 of hCATL A, which together serve as an intron for hCATL AIII. HCATL AIII was observed to be the most abundant splice variant in five different human cell lines. In vitro transcription coupled translation studies revealed that hCATL AIII is translated with 4.4-, 3.9- and 1.6-fold higher efficiency as compared to hCATL A, AI and AII, respectively. These results were further confirmed by measuring the enzymatic activities of the in vitro translated products. Cloning of hCATL AIII UTR upstream to luciferase reporter gene resulted in a 3.75-fold higher expression of the reporter gene as compared to the luciferase construct containing UTR of hCATL A. Thus, we have identified a novel human cathepsin L splice variant, hCATL AIII, which is most abundant in human cell lines and is translated with highest efficiency. Our results demonstrate either the presence of a positive or absence of a negative cis-acting regulatory element(s) in the UTR of hCATL AIII that is sufficient to confer translational advantage to a heterologous mRNA. The predominance of this most efficiently translated splice variant in malignant cells suggests that it plays a key role in the over-expression of human cathepsin L in cancer.