Tissue-specific alternative splicing of spermidine/spermine N1-acetyltransferase.
ABSTRACT The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N(1)-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N(1),N(11)-diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.
Article: Duplication and Diversification of the Spermidine/Spermine N(1)-acetyltransferase 1 Genes in Zebrafish.[show abstract] [hide abstract]
ABSTRACT: Spermidine/spermine N(1)-acetyltransferase 1 (Ssat1) is a key enzyme in the polyamine interconversion pathway, which maintains polyamine homeostasis. In addition, mammalian Ssat1 is also involved in many physiological and pathological events such as hypoxia, cell migration, and carcinogenesis. Using cross-genomic bioinformatic analysis in 10 deuterostomes, we found that ssat1 only exists in vertebrates. Comparing with mammalian, zebrafish, an evolutionarily distant vertebrate, contains 3 homologous ssat1 genes, named ssat1a, ssat1b, and ssat1c. All zebrafish homologues could be transcribed and produce active enzymes. Despite the long history since their evolutionary diversification, some features of human SSAT1 are conserved and subfunctionalized in the zebrafish family of Ssat1 proteins. The polyamine-dependent protein synthesis was only found in Ssat1b and Ssat1c, not in Ssat1a. Further study indicated that both 5' and 3' sequences of ssat1b mediate such kind of translational regulation inside the open reading frame (ORF). The polyamine-dependent protein stabilization was only observed in Ssat1b. The last 70 residues of Ssat1b were crucial for its rapid degradation and polyamine-induced stabilization. It is worth noting that only Ssat1b and Ssat1c, but not the polyamine-insensitive Ssat1a, were able to interact with integrin α9 and Hif-1α. Thus, Ssat1b and Ssat1c might not only be a polyamine metabolic enzyme but also simultaneously respond to polyamine levels and engage in cross-talk with other signaling pathways. Our data revealed some correlations between the sequences and functions of the zebrafish family of Ssat1 proteins, which may provide valuable information for studies of their translational regulatory mechanism, protein stability, and physiological functions.PLoS ONE 01/2013; 8(1):e54017. · 4.09 Impact Factor