Alternative splicing of type II procollagen exon 2 is regulated by the combination of a weak 5' splice site and an adjacent intronic stem-loop cis element.
Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Chondroprogenitor cells produce the type IIA procollagen isoform by splicing (including) exon 2 during pre-mRNA processing, whereas differentiated chondrocytes synthesize the type IIB procollagen isoform by exon 2 skipping (exclusion). Using a COL2A1 mini-gene and chondrocytes at various stages of differentiation, we identified a non-classical consensus splicing sequence in intron 2 adjacent to the 5' splice site, which is essential in regulating exon 2 splicing. RNA mapping confirmed this region contains secondary structure in the form of a stem-loop. Mutational analysis identified three cis elements within the conserved double-stranded stem region that are functional only in the context of the natural weak 5' splice site of exon 2; they are 1) a uridine-rich enhancer element in all cell types tested except differentiated chondrocytes; 2) an adenine-rich silencer element, and 3) an enhancer cis element functional in the context of secondary structure. This is the first report identifying key cis elements in the COL2A1 gene that modulate the cell type-specific alternative splicing switch of exon 2 during cartilage development.
McAlinden
Audrey
A
Department of Orthopaedic Surgery, Washington University School of Medicine, St Louis, Missouri 63110, USA. amcalind@msnotes.wustl.edu
Havlioglu
Necat
N
Liang
Li
L
Davies
Sherri R
SR
Sandell
Linda J
LJ
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D019902:Chondrocytes / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics*; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005091:Exons; D006801:Humans; D007438:Introns; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011347:Procollagen / Q000235:genetics*; D020033:Protein Isoforms; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013234:Stem Cells; D014162:Transfection; D014529:Uridine / Q000737:chemistry
C499177:COL2A1 protein, human; D024043:Collagen Type II; D017931:DNA Primers; D011347:Procollagen; D020033:Protein Isoforms; D012333:RNA, Messenger; D012313:RNA; D014529:Uridine