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

2003 ◽  
Vol 18 (9) ◽  
pp. 1716-1722 ◽  
Author(s):  
Takayuki Nishiyama ◽  
Hiroshi Hatano ◽  
Masahiro Kurosaka ◽  
Mark E Bolander ◽  
Gobinda Sarkar
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Exon 2 ◽  
Cis Acting ◽  
Specific Alternative

scholarly journals 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

2005 ◽  
Vol 280 (38) ◽  
pp. 32700-32711 ◽  
Author(s):  
Audrey McAlinden ◽  
Necat Havlioglu ◽  
Li Liang ◽  
Sherri R. Davies ◽  
Linda J. Sandell
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Type Ii ◽  
Stem Loop ◽  
Exon 2 ◽  
Cis Element

Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence.

Genetics ◽  
1992 ◽  
Vol 131 (1) ◽  
pp. 113-128 ◽  
Author(s):  
M T O'Neil ◽  
J M Belote
Keyword(s):  
Alternative Splicing ◽  
Drosophila Virilis ◽  
Evolutionary Divergence ◽  
Sequence Comparisons ◽  
Specific Expression ◽  
Cis Acting ◽  
Conserved Sequence ◽  
Homologous Genes ◽  
Specific Alternative ◽  
High Degree

Abstract The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 815-815
Author(s):  
Marilyn Parra ◽  
Xiuli An ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Start Codon ◽  
Exon 2 ◽  
Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint

2001 ◽  
Vol 355 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Anna-Marja K. SÄÄMÄNEN ◽  
Heli J. SALMINEN ◽  
A. Juho RANTAKOKKO ◽  
Dick HEINEGÅRD ◽  
Eero I. VUORIO
Keyword(s):  
Genomic Structure ◽  
Type Ii Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Primary Role ◽  
Type Ii ◽  
Exon 2 ◽  
Collagen Fibrillogenesis ◽  
Calcified Cartilage ◽  
Age Related

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7kb long cDNA contains an open reading frame of 1131nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.

Expression of type II procollagens during development of the human intervertebral disc

2002 ◽  
Vol 30 (6) ◽  
pp. 831-838 ◽  
Author(s):  
A. McAlinden ◽  
Y. Zhu ◽  
L. J. Sandell
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type Ii Collagen ◽  
Intervertebral Discs ◽  
Bone Morphogenetic Protein 2 ◽  
Type Ii ◽  
Exon 2 ◽  
Morphogenetic Protein ◽  
Alternatively Spliced ◽  
Specific Stage

Mice lacking type II collagen fail to develop intervertebral discs. The present study describes the distribution of the developmentally expressed type IIA procollagen molecule, as well as types I and III collagens, in human IV disc specimens ranging from 42 to 101 days gestation. Type IIA procollagen contains the alternatively spliced exon 2 which encodes a 69-amino-acid cysteinerich domain. By radioactive in situ hybridization and fluorescence immunohistochemistry, we identified changes in the localization patterns of type IIA procollagen, particularly between days 54 and 101. At day 54, the developing disc was divided into the outer annulus containing types I and III collagens, the inner annulus containing type IIA procollagen and the notochord consisting of all three fibrillar collagens. Specifically, the IIA N-terminal propeptide was localized in the extracellular matrix at day 54 but, by day 101, was only observed in the cytoplasm of the inner annulus cells. A functional role for the IIA N-terminal propeptide during this specific stage of disc development seems apparent. This function may involve regulation of growth factors since the exon 2-encoded domain of type IIA procollagen has previously been shown to bind to bone morphogenetic protein-2 and transforming growth factor-β. We aim to explore this mechanism further.

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