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RAT FOLLISTATIN-RELATED GENE (FLRG): THE DEDUCED AMINO-ACID SEQUENCE AND EXPRESSION IN FEMALE REPRODUCTIVE AND NON-REPRODUCTIVE TISSUES.
Arai, Koji1, Tsuchida, Kunihiro2, Uehara, Kohkichi1, Taya, Kazuyoshi3, Sugino, Hiromu2, 1 2 3
ABSTRACT- Follistatin-related gene (FLRG) was originally identified as a target of a t(11;19)(q13;p13) chromosomal translocation in a human B-cell leukemia. FLRG protein is structurally similar to follistatin. Therefore, FLRG is postulated as a new TGF-beta superfamily binding protein, which may modulate actions of TGF-beta superfamily proteins. Until now, human and mouse FLRG cDNAs have been cloned, and recombinant mouse FLRG fusion proteins proved to bind to activin A and BMP-2. However, to elucidate the roles of FLRG in reproductive events, characterization of rat FLRG would be required because the rat is one of the most widely used experimental animals for study of reproduction. In this study, we report a complete amino acid sequence of rat FLRG protein and expression of FLRG mRNA in reproductive and non-reproductive tissues. The rat FLRG cDNAs were cloned by PCR-based methods, 5' and 3' RACE (rapid amplification of cDNA ends). Placentae collected on day 13 of pregnancy were used as the sources of the template cDNAs. The RACE products revealed that rat FLRG mRNA has a 0.8kb coding region and a 1.1kb 3' untranslated region. Northern blot hybridization showed that rat FLRG mRNA was 2.0kb in length, and the size is consistent with the RACE products. The deduced rat FLRG protein consists of 256 amino acid residues as well as mouse FLRG, whereas human FLRG composed of 263 amino acid residues. The amino acid sequence of rat FLRG shares 97 and 82 % homology with mouse and human FLRG, respectively. RNase protection assay using digoxygenin (DIG)-labeled RNA probes showed that rat FLRG mRNA was abundantly expressed in the placenta, whereas follistatin mRNA was undetectable or detected at very low levels in the tissue. Rat FLRG was also expressed in other reproductive tissues such as the ovary and uterus, and in non-reproductive tissues such as the lung and adrenal gland. The distribution of rat FLRG mRNA was different from the distribution of follistatin mRNA, suggesting that these proteins differently regulate physiological events.
KEY WORDS: follistatin, FLRG, rat