Cis-acting intronic elements that regulate cartilage-specific alternative splicing of the type II collagen (Col2) pre-mRNA lie at or near splice site junction sequences flanking exon 2 of the gene.
Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene.


BACKGROUND
Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors.


METHODS
To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction.


RESULTS
The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14-21 days) with insulin.


CONCLUSIONS
These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.
Nishiyama
Takayuki
T
Department of Orthopedic Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Hatano
Hiroshi
H
Kurosaka
Masahiro
M
Bolander
Mark E
ME
Sarkar
Gobinda
G
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002356:Cartilage / Q000378:metabolism*; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics*; D004247:DNA / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D005816:Genetic Complementation Test; D007328:Insulin / Q000494:pharmacology; D007438:Introns; D051379:Mice; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion
D024043:Collagen Type II; D007328:Insulin; D012322:RNA Precursors; D004247:DNA