Tissue-specific alternative splicing of spermidine/spermine N (1)-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.
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ABSTRACT: Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX) that is involved in SAT1 regulated unproductive splicing and transcription (RUST), are involved. We applied next generation sequencing (RNA-seq) to assess gene-level, isoform-level, and exon-level SAT1 expression differences between healthy controls (HC, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9, BA9) of medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA species putatively involved in SAT1 post-transcriptional regulation. A DSM-IV diagnosis was made by structured interview. Toxicology and history ruled out recent psychotropic medication. At the gene-level, we found low SAT1 expression in both MDD-S (vs. HC, p=0.002) and MDD (vs. HC, p=0.002). At the isoform-level, reductions in MDD-S (vs. HC) were most pronounced in four transcripts including SSAT and SSATX, while reductions in MDD (vs. HC) was pronounced in three transcripts, one of which was reduced in MDD relative to MDD-S (all p<0.1 FDR corrected). We did not observe evidence for differential exon-usage (i.e. splicing) nor differences in miRNA expression. Results replicate the finding of low SAT1 brain expression in depressed suicides in an independent sample and implicate low SAT1 brain expression in MDD independent of suicide. Low expression of both SSAT and SATX isoforms suggest shared transcriptional mechanisms involved in RUST may account for low SAT1 brain expression in depressed suicides. Future studies are required to understand the functions and regulation of SAT1 isoforms, and how they relate to the pathogenesis of MDD and suicide. Copyright © 2015. Published by Elsevier Inc.Neurobiology of Disease 05/2015; 79. DOI:10.1016/j.nbd.2015.04.014 · 5.20 Impact Factor
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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. DOI:10.1371/journal.pone.0054017 · 3.53 Impact Factor
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ABSTRACT: Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST).PLoS Genetics 10/2013; 9(10):e1003743. DOI:10.1371/journal.pgen.1003743 · 8.17 Impact Factor