Abstract
Abstract: :
Purpose: The large tumor suppressor gene (lats1) encodes a highly conserved (from fly to human) protein kinase that plays a crucial role in the prevention of tumor formation by controlling the progression of mitosis. Previous studies revealed that the mRNA corresponding to lats1 is present in most tissues as a 7.5 kb transcript. We have found that in addition to this long form of lats1 (lats1L) mRNA, a less abundant, shorter 3.4 kb primary transcript (lats1S) is expressed in the vertebrate retina. The aim of the current study was to characterize the lats1S cDNA and its gene product. Methods: A fragment of the lats1S cDNA was isolated by cDNA representational difference analysis (RDA). Three rounds of subtractive hybridization were performed to obtain a difference product. Subtracted RDA products were ligated to pBluescript KS II+ and analyzed. We used a polyclonal peptide antiserum, NK1, generated against the N-terminal region (18 aa) of lats1 to examine the retinal expression pattern of lats1 proteins. Results: The nucleotide sequence of the complete lats1S cDNA, isolated from a retinal cDNA library, revealed that this mRNA is generated by alternative splicing of the lats1 gene. The 3' UTR sequences of both lats1 transcripts are different. The lats1S cDNA encodes an isoform of the putative Ser/Thr protein kinase lats1. This isoform has seven highly conserved motifs at the C-terminus which constitute the catalytic domain, instead of the eleven motifs that are present in most members of the eukaryotic protein kinase superfamily. Western-blot analysis of retinal extracts detected two distinct bands with molecular masses corresponding to both the lats1S and lats1L proteins. The size of these proteins were found to be 170 kDa and 120 kDa, respectively. Conclusion: The expression of both long and short lats1 isoforms in vertebrate retinal cells raises the possibility that these lats1 proteins may act as negative key regulators of the cell cycle each of them performing a unique role. Since the function of the retinally expressed lats1S protein is unknown, we see the merit for further studies of this protein.
Keywords: 417 gene/expression • 476 molecular biology • 554 retina