Developmentally regulated alternative splicing of the alpha1(XI) collagen chain: spatial and temporal segregation of isoforms in the cartilage of fetal rat long bones.

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the alpha1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of alpha1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.     

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the alpha1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices.

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of alpha1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express alpha1(XI), as well as 293 cells which do not express alpha1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of alpha1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.     

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the α1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of α1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express α1(XI), as well as 293 cells which do not express α1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of α1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.     

Expression, purification, and refolding of recombinant collagen alpha1(XI) amino terminal domain splice variants

The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.     

Assignment of the human collagen alpha 1 (XIII) chain gene (COL13A1) to the q22 region of chromosome 10

Type XIII collagen is a recently described collagen that resembles in structure the short-chain collagens of types IX, X, and XII. Unlike any other collagen, the type XIII is found in several different forms generated through alternative splicing. A 2.0-kb genomic fragment from the human alpha 1 (XIII) collagen gene was isolated and shown by DNA sequencing to contain exon 12 as counted from the 3' end. This fragment was used as a probe to localize the gene. The gene (COL13A1) was assigned to chromosome 10 by hybridization of the probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes. Furthermore, the gene was mapped to the q22 region by in situ hybridization to metaphase chromosomes.     

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1^+ ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1^+ ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.     

Mapping of a human fibrillar collagen gene, pro alpha 1 (XI) (COL11A1), to the p21 region of chromosome 1

Type XI collagen is a minor and poorly characterized structural component of cartilage. Recently, cDNA and genomic clones coding for the pro alpha 1 chain of human Type XI collagen, formerly 1 alpha collagen, have been isolated and fully characterized. Here we have used one such probe to establish the chromosomal localization of the pro alpha 1 (XI) collagen gene (COL11A1) by hybridization to filter-bound DNA isolated from flow-sorted chromosomes and by in situ hybridization on metaphase chromosomes. This combination of approaches has enabled us to locate COL1A11 in the p21 region of chromosome 1. This represents the first mapping of a Type XI collagen gene and the first assignment of a collagen locus to chromosome 1. These studies also provide additional evidence for the nearly uniform dispersion of the human fibrillar collagen genes in the human genome.     

Multiple isoforms of beta-TrCP display differential activities in the regulation of Wnt signaling

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.     

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene ( KLC1 ) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.