| Runx2 ( also known as Cbfal, Osf2, PEBP2aA, and AML3) is a complex gene in which two promoters, P1 and P2, transcribe two major isoforms, designated here as Runx2-II and Runx2-I, that differ in their respective 5' untranslated regions (respectively designated UTR1 and UTR2) and N-termini but are identical with respect to their DNA-binding domains and C-termini, which are encoded by exons 2-8. Runx2-II (also called major til-1, Cbfal.iso or Cbfal/p57) is transcribed from the distal P1 promoter and encodes a 528 amino acid protein that begins with the 19 amino acids MASNSLFSAVTPCQQSFFW derived from exon la. Runx2-I (also called PEBP2alphaA, Cbfal/org or Cbfal/p56) is transcribed from the proximal P2 promoter and encodes a 514 amino acid protein that begins with the 5 amino acids MRIPV located in exon 1b. Although the Runx2 gene encodes two major osteoblast-specific transcription factors that regulate osteoblast differentiation, uncertainty exists about the structure of their 5' end and expression in osteoblasts, various cell lines and different tissues.In the current study, we have characterized the genomic structure of the human, rat, and mouse 5' Runx2-II sequence, and evaluated the expression of two major isoforms in osteoblasts, cell line and multiple tissue panels. We found the novel 5' Runx2-II sequence (exon la) is highly conserved across species that the overall sequence identity of mouse, rat and human sequences was 95% and between rat and mouse 97%. Mouse Exon la is divided into two parts by a 165 bp mini-intron, and has at least two splice donor sites (sd1 and sd2) in the 5'UTR1 and one additional splice donor site (sd3) in the coding region of exonla. Alternative splice site (sd1 or sd2) selection generates additional transcripts, Runx2-IId1 and Runx2-IId2 that differ by 33 nucleotides in the 5' UTR1 of Runx2-II. Using Exon la specific primer pair and mRNA purified from MC3T3-E1, we obtained and sequenced three RT-PCR products, including unspliced transcripts UTRlu, UTRld1 and UTRld2. Based on the similarity of the deduced amino acid sequence among the mouse, rat and human Runx2-IIisoforms encoded by Exon la, the strong Kozak consensus sequence around ATG1 and the absence of the unique OSF2 transcript in human suggests that the RUNX2/OSF2 isoform begins with MASNSL, rather than the novel N-terminal OSF2 sequence (MLHSPH). Primer extension mapping identified in the Runx2-II P1 promoter rigion a minor mRNA start site located -0.8 kb 5' upstream of the ATG1 encoding the Runx2-II (MASNS) isoform and -0.4 kb upstream of the previously reported start site.Northern blot analysis with Runx2 exon 1a and exon 2 probe revealed that both MC3T3E1 and ROS17/2.8 osteoblasts expressed two major transcripts of 6.0 and 5.4 kb. Further analysis with Runx2 exon la probe only detected the lower 5.4-kb band (Runx2-II), whether the upper 6.0-kb band represented Runx2-I or other Runx transcripts need to be confirmed by Runx2 exon 1b probe. In addition, RT-PCR cloning analysis identified Runx2-containing transcripts in mouse osteoblasts with internal deletions due to skipping of either exon 2, exon 5, or both exons 2 and 5. Thus, multiple minor and smaller transcripts were also observed in the same northern blot, which might correspond to the transcripts via selective loss of exon 2 and 5.To clarify the cell-type specificity of Runx2 expression, we performed RT-PCR, RNase protection analysis and real-time PCR of total RNA derived from several different cell lines using Runx2-I and Runx2-II specific primers and a Runx2- II specific riboprobe (239, 465). We observed that all except Jurket cells expressed Runx2-I, but others except non-osteoblastic Hepal-6 and NIH3T3 cells, including Jurket, pluripotent C3H10T1/2, C2C12, chondrocyte C5.18 and osteoblasts MC3T3E1, UMR106, ROS17/2.8, expressed Runx2-II. RNase protection identified two bands of 227 and 124 bp in osteoblasts MC3T3E1 and ROS17/2.8, consistent with a transcript containing the "mini-intron" in the 5' UTR (Runx2-IIu) and a mRNA species in which this "mini-int... |
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