Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/pseudogenes.

Abstract

Mouse SMT3A and SMT3B cDNAs encoding ubiquitin-like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced. The sequence of the first 92 amino acids (ending with the conserved Gly-Gly) of mouse SMT3A exhibited two differences at amino acid no. 38 and 76 in comparison with that of human SMT3A. The C-terminal 18 amino acid sequence of mouse SMT3A was completely different from the C-terminal 11 amino acid sequence of human SMT3A. Mouse and human SMT3B were identical for a sequence of 95 amino acids. Mouse SMT3A genomic DNAs were amplified by polymerase-chain-reaction and sequenced. The nucleotide sequence of a PCR-amplified SMT3A genomic DNA fragment was found to be identical to that of SMT3A cDNA, indicating the absence of intron(s) in its protein coding region. Another genomic DNA fragment of 1,531 nucleotides, containing 7% differences from that of cDNA, is unable to encode a functional protein, and thus, it is a SMT3A processed pseudogene. Three mouse SMT3B processed pseudogenes were cloned and sequenced. The genuine mouse SMT3B gene has not yet been isolated. Mouse SMT3A transcript of 1.8 kb was predominantly expressed in most tissues, while SMT3B transcript of 1.0 kb was abundantly present in all tissues analyzed. A family of ubiquitin-like proteins was recently discovered. One distinguishing feature of ubiquitin and ubiquitin-like proteins is the capacity to conjugate with other proteins post-translationally. The ubiquitin-like proteins are cleaved endoproteolytically after a diglycine sequence, corresponding to the C-terminal Gly75-Gly76 of ubiquitin. The cleavage activates the molecule for conjugation. The yeast SMT3 gene was originally identified as a suppressor of mutations in MIF2 gene, which encodes an essential protein binding to the A+T-rich CDEII region of centromere DNA (1). Studies using temperature-sensitive mutants showed that the loss of yeast Mif2 protein function results in chromosome missegregation, mitotic delay, and aberrant microtubule morphologies (2). The yeast Mif2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C, an integral component of active kinetochores (3, 4). Human SMT3A cDNA was identified from the genome sequencing project of chromosome 21 (5). We have cloned human SMT3B (formerly designated as HSMT3) cDNA (6). Human SMT3C protein was independently isolated by several groups and denoted as SUMO-1 (7), GMP1 (8), PICI (9), UBL1 (10), sentrin (11). SUMO-1/GMP1 was found to be covalently linked to the Ran GTPase-activating protein RanGAP1, and attachment of SUMO-1 targets the otherwise cytosolic RanGAP1 to the nuclear pore complex. The modified form of RanGAP1 also appeared to associate with the mitotic spindle apparatus during mitosis (7, 8). PIC1 was shown to interact with the PML component of nuclear multiprotein complex that is disrupted in acute promyelocytic leukemia (9). UBL1 was found to associate with human RAD51/RAD52 proteins involved in DNA recombination and DNA double-strand break repair (10). Sentrin was shown to interact with Fas/APO-1 or the TNF receptor 1 death domain, and the overexpression of sentrin provided protection against both anti-Fas/APO-1 and TNF-induced cell death (11). Here we report the characterization of mouse SMT3A and SMT3B cDNAs, gene/pseudogenes, and mRNA expression.