Identification and analysis of human RCAN3 (DSCR1L2) mRNA and protein isoforms.
Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, "reference" isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.
Facchin
Federica
F
Department of Histology, Embryology and Applied Biology, University of Bologna, Via Belmeloro, Bologna (BO), Italy.
Canaider
Silvia
S
Vitale
Lorenza
L
Frabetti
Flavia
F
Griffoni
Cristiana
C
Lenzi
Luca
L
Casadei
Raffaella
R
Strippoli
Pierluigi
P
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D020107:Troponin T / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D020033:Protein Isoforms; D011506:Proteins; C408372:RCAN3 protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C518660:TNNT3 protein, human; D020107:Troponin T; D005982:Glutathione Transferase