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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.
J Bone Miner Res. 2003 Sep; 18(9):1716-22.JB

Abstract

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.

INTRODUCTION

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.

MATERIALS AND 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.

CONCLUSION

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.

Authors+Show Affiliations

Department of Orthopedic Research, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12968682

Citation

Nishiyama, Takayuki, et al. "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." Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, vol. 18, no. 9, 2003, pp. 1716-22.
Nishiyama T, Hatano H, Kurosaka M, et al. 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. J Bone Miner Res. 2003;18(9):1716-22.
Nishiyama, T., Hatano, H., Kurosaka, M., Bolander, M. E., & Sarkar, G. (2003). 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. Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, 18(9), 1716-22.
Nishiyama T, et al. 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. J Bone Miner Res. 2003;18(9):1716-22. PubMed PMID: 12968682.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Nishiyama,Takayuki, AU - Hatano,Hiroshi, AU - Kurosaka,Masahiro, AU - Bolander,Mark E, AU - Sarkar,Gobinda, PY - 2003/9/13/pubmed PY - 2004/5/1/medline PY - 2003/9/13/entrez SP - 1716 EP - 22 JF - Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research JO - J Bone Miner Res VL - 18 IS - 9 N2 - UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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. CONCLUSION: 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. SN - 0884-0431 UR - https://www.unboundmedicine.com/medline/citation/12968682/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_ L2 - https://doi.org/10.1359/jbmr.2003.18.9.1716 DB - PRIME DP - Unbound Medicine ER -