©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternative mRNA Processing Occurs in the Variable Region of the Pro-1(XI) and Pro-2(XI) Collagen Chains (*)

Natalia I. Zhidkova , Susan K. Justice , Richard Mayne (§)

From the (1) Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An analysis was performed of differential splicing of primary transcripts in the noncollagenous variable region located in the amino terminus of the pro-1(XI) and pro-2(XI) collagen chains. The results for the pro-2(XI) chain showed that human cartilage or fibroblasts in culture contain transcripts in which a single highly acidic exon encoding for 21 amino acids is present or absent. For the chicken pro-1(XI) chain a more complex pattern of alternative splicing was detected with six possible variants. Of special interest was the alternative use of two exons (called IIA and IIB) in which IIA encodes for 39 amino acids and is highly acidic (estimated pI = 3.2), whereas IIB encodes for 49 amino acids and is highly basic (estimated pI = 10.6). A similar alternative use of exon IIA or exon IIB was also observed for human chondrocytes. Northern blotting with probes specific for IIA or IIB showed that both exons are present in transcripts from cartilage but exon IIB is preferentially utilized in transcripts from tendon. Present results suggest that both the pro-1(XI) and pro-2(XI) chains of type XI collagen undergo limited processing in vivo and that the noncollagenous variable region is initially retained on the surface of the fibrils. Differential splicing in the variable region may potentially influence the interaction of collagen fibrils with other molecules of the extracellular matrix and more specifically with sulfated glycosaminoglycan chains or with hyaluronan. Such interactions may play a key role in establishing both the organization of the collagen fibrils within the extracellular matrix and in limiting the diameter of collagen fibrils.


INTRODUCTION

In many tissues, such as cornea or vitreous humor, the diameter of each collagen fibril is carefully regulated (1) . However, the mechanism by which this is controlled is still unknown. Numerous studies have shown that most, if not all, collagen fibrils contain a small proportion of one or more members of the type V/XI family of collagens (2, 3, 4, 5, 6) . Originally, it was thought that type XI collagen is specific for cartilaginous tissues (3) . However, other studies have demonstrated the presence of transcripts for both the pro-1(XI) chain (7, 8, 9) and pro-2(XI) chain (10, 11) in a variety of cells in culture and in different tissues during development. In addition, adult bovine bone was shown to contain a significant amount of the 1(XI) chain in addition to the 1(V) chain (12) . More recent results show that a molecule of chain organization 1(XI)2(V) is the major collagen synthesized by the rhabdomyosarcoma cell line A204 (13) . In addition, the isolation of 1(XI) and 2(V) chains, but not 2(XI) chains, from bovine vitreous (6) , further supports the growing evidence that there is a single type V/XI family of collagens with a variety of different chain organizations (14) . It appears likely that all of the collagen molecules in this family will share a common function, and that individual members of the family may be potentially involved in the generation of tissue-specific collagen fibrils.

Initially, a function for the type V/XI collagen family was suggested from in vitro experiments which demonstrated that type V collagen will limit the diameter of collagen fibrils formed from type I collagen (15, 16) . Similar experiments indicating an involvement of type XI in the control of fibril diameter were reported for cartilage collagens in which type II, type IX, and type XI collagen were all found to be required for the assembly of fibrils of constant diameter (17) . Separate evidence for an involvement of the type V/XI collagen family in influencing fibril diameter was obtained from an analysis of the cho/cho mouse which exhibits an autosomal recessive chondrodysplasia in which thick collagen fibrils are observed by electron microscopy within the cartilage matrix (18, 19) . Recent analyses show that the cho mutation is located in the mouse 1(XI) collagen gene and that these animals have only a limited ability to synthesize the pro-1(XI) chain (20) . In addition, two Dutch families were recently analyzed each with an inherited chondrodysplasia resembling Stickler syndrome but without eye involvement. In both families mutations in the 2(XI) chain were located showing the importance of type XI collagen in the cartilagenous structures of man (21) .

A model was presented recently for the location of type V on the surface of a fibril of type I collagen present in the chicken embryonic cornea (22) . It was proposed that the major collagen triple helix of type V collagen lies buried within the fibril (as was also proposed earlier (4, 23) ). However, the amino terminus of each molecule, including a second short collagen triple helix, was proposed to project away from the surface of the fibril. Thus, the noncollagenous amino terminus of each molecule could potentially interact with other connective tissue macromolecules such as proteoglycans, and therefore be involved in maintaining the overall organization of the matrix. The nucleotide-derived amino acid sequence of the amino terminus of the human and chicken 1(V) chain (22, 24, 25) , the human 2(V) chain (26) , the human and bovine 1(XI) chain (8, 27) , and the human 2(XI) chain (28) are all available. Analysis of these sequences shows that the pro-2(V) chain is closely related to the chains of type I, II, and III collagen, whereas the pro-1(V), pro-1(XI), and pro-2(XI) chains are closely related to each other and share several unique features (14, 25, 28) . For the pro-2(XI) chain, a disulfide-bonded peptide called proline/arginine-rich protein or PARP() is released from the amino terminus during extracellular processing and is subsequently retained in the cartilage matrix from which it can be isolated in significant amounts (29) . The pro-1(V) and pro-1(XI) chains also have an amino-terminal domain which is homologous in amino acid sequence and location of cysteine residues to PARP (30-32; see Fig. 1). However, it is not known if there is a specific cleavage and release of these PARP-like domains into the matrix. For the pro-1(V), pro-1(XI), and pro-2(XI) chains there is a highly acidic amino acid sequence which follows the basic PARP-like domains and is variable in length. This is the only region of the molecule for which little or no homology can be recognized in amino acid sequence when the 1(V), 1(XI), and 2(XI) chains are compared (24, 25, 28) . We previously called this sequence the variable region (28) . We have now performed a detailed analysis of transcripts containing the variable region using RT-PCR and, with additional analyses of the genomic organization, report alternative mRNA processing of the pro-1(XI) and pro-2(XI) chains.


Figure 1: Model for the domain structure of the pro-2(XI), pro-1(XI), pro-1(V), and pro-2(V) chains. The order of the domains is as described previously (28) with COL1 forming the major collagen triple helix and COL2 forming the minor, amino-terminal triple helix. Note that there are three noncollagenous domains (NC1 NC3) and that the NC3 domain is further subdivided into the signal peptide ( SP), the PARP region, the variable region ( VR), and a short constant region ( CR), the latter being highly homologous in primary amino acid sequence for all three chains. Also note that the pro-2(V) chain does not contain a PARP-like domain, a variable region, or a constant region.




MATERIALS AND METHODS

Sources of Cells and Tissues

Human chondrocytes were isolated from sternal cartilage obtained from an 8-year-old white female during pectus excavatum surgery as described previously (28) . Human skin fibroblasts were initially obtained from a skin biopsy of a 34-year-old white female and were at the fifth passage. The cells were grown in McCoy's modified 5a medium plus 10% fetal calf serum and 1% antibiotic antimycotic. Human gingival fibroblasts were obtained as outgrowths from a normal adult male and grown in minimal essential medium ( medium) plus 10% fetal calf serum, 1% L-glutamine, and 4% antibiotic/antimycotic mixture. The cells were passaged with trypsin four to six times before being utilized for RNA isolation. Human histiocytic lymphoma cells were obtained from American Type Culture Collection (ATCC CRL 1593 U-937) and were grown in Dulbecco's minimal essential medium containing 10% fetal calf serum and 1% antibiotic/antimycotic. Chicken chondrocytes were obtained from the sterna of 17-day embryos and were isolated as described previously (33) . All culture medium and supplements were from Life Technologies, Inc./BRL Life Technologies, Gaithersburg, MD.

Chicken gizzard, heart, kidney, skeletal muscle, brain, skin, lung, liver, and tendon were all dissected from 17-day embryos and used to isolate RNA without dissociation into individual cells.

Isolation of RNA and First Strand cDNA Synthesis

Total RNA from cells or tissues was obtained by the single step acid-guanidine method (34) and poly(A)-rich RNA selected by oligo(dT)-cellulose chromatography (35) . Synthesis of first strand cDNA was based on a kit (Reverse Transcription System; Promega Corp., Madison, WI) using either 5 µg of poly(A)-rich RNA or 10-30 µg of total RNA in 50 µl of buffer. A mixture of random primers (10 µl) was added and the mixture incubated at 65 °C (10 min) and rapidly cooled on ice (1 min). To the RNA/primer mixture, 5 µl of 100 m M methylmercuric hydroxide (Alfa/Johnson Matthey, Ward Hill, MA) was added with incubation for 10 min at room temperature followed by 5 µl of 700 m M -mercaptoethanol and incubation for 5 min at room temperature. For first strand cDNA synthesis, avian myeloblastosis virus reverse transcriptase (Promega) was employed with incubation at 45 °C for 45 min followed by 55 °C for 15 min. Precipitation was performed with absolute ethanol in the presence of 3 M Na-acetate, pH 5.2 (10:1 ratio), at -20 °C and the pellet washed with ice-cold ethanol (80%).

Total RNA was isolated from 17-day chick chondrocytes and other chick tissues using RNAzolB according to the manufacturer's instructions (Biotecx Laboratories, Inc., Houston, TX). Poly(A)-rich RNA was isolated with streptavidin-paramagnetic particles using a kit (PolyATract mRNA Isolation System; Promega). First strand cDNA was prepared from chicken tissues as described above for human cells.

Polymerase Chain Reaction (PCR)

In order to analyze the variable region of the human pro-2(XI) chain (28) , PCR was performed with primers DE1 (5`-CAGAAGGAGCTGGAATGCGAG-3`) and PO2 (5`CGGAATGGGAGCATGAGAGA3`) using GeneAmp PCR core reagents (Perkin Elmer, Norwalk, CT), and Taq I polymerase (Promega). The cycle parameters were 2 min denaturation (94 °C), 1.5 min annealing (58 °C), and 3 min polymerization (72 °C) for 33 cycles with 5 min final elongation (75 °C) using a Programmable Thermal Controller (MJ Research Inc., Watertown, MA). For the initial PCR to analyze the variable region of the chicken pro-2(XI), pro-1(XI), and pro-1(V) chains, degenerate oligonucleotides XB1 5`-GA(CT)GA(AG)GA(AG)GTITT(CT)GA(AG)GG-3` and XB2 5`-GG(TC)TCICC(TC)TT(TC)T(GC)(TGCA)CC(TC)TT-3` were designed from shared amino acid sequences with substitution by inosine residues at some sites in the 5` ends of the primers (33) . Subsequently, the variable region of the chicken pro-1(XI) chain was analyzed by RT-PCR with primers PL1 and PL2 located in exons I and V (see Fig. 6 for location and sequence of primers).


Figure 6: Nucleic acid sequence of the variable region of the chicken pro-1(XI) chain with a comparison to the human pro-1(XI) chain. Location of several primers (sense and antisense) is shown by underlining the nucleotides. Intron/exon boundaries are shown by . The nucleic acid sequence and derived peptide sequence for human I, IIA, III, IV, and V was taken from Ref. 27. The nucleic acid sequence of the previously undescribed human exon IIB is also included in the figure. Primers XB1 and XB2, location of the degenerate primers used initially to amplify the differentially spliced variants. Primers PL1 and PL2 were used to investigate the differentially spliced variants specifically of the chicken pro-1(XI) chain. Primers VS4 and VS5 were used to amplify specifically exon IIA and primers VC1 and VC2 were used to amplify exon IIB.



Screening of Genomic Libraries

For isolation of a human genomic 2(XI) clone, a human leukocyte genomic library constructed in EMBL 3A vector (Clontech Laboratories, Inc., Palo Alto, CA) was used. For screening, a 162-bp fragment was generated by PCR on the NZ2 clone derived from human chondrocyte RNA (28) using primers LA5 = 5`-TCAAGACTTCACAGGCCACA-3` and LA6 = 5`-TTCCAACACTGCAGGCTCTC-3`. The 162-bp fragment was extracted from agarose gel using a QIAEX agarose gel extraction kit (Qiagen Inc., Chatsford, CA). For screening, the 162-bp fragment was labeled using a random primed DNA labeling kit (Boehringer Mannheim). The specific activity of labeling was ≅1 10cpm/µg DNA. The labeling reaction was performed with 3000 mCi/mmol [-P]dCTP (DuPont NEN).

Two overlapping genomic clones were obtained (HGCol18 and HGCol9). For sequencing, the whole insert was first removed by digestion with HindIII or EcoRI/ SacI, and then subcloned from the HGCol18 clone into pBluescript II SK+ plasmid. The constructed plasmids were called p18H15 and p18RS12 and used for DNA sequencing and analysis of genomic structure.

For the isolation of chicken genomic 1(XI) clones a chicken genomic library in FIX II vector (Stratagene) was screened with 558- and 528-bp fragments generated by PCR on plasmid DNAs containing exon IIB (called pXB28) or exon IIA (called pXB45) using primers PL1 and PL2 (see Fig. 6). Fragments were labeled using a random primed DNA labeling kit (Boehringer Mannheim). Two overlapping genomic clones (CHCol5 and CHCol13) were obtained. The whole inserts from both clones were subcloned into pBluescript II using the NotI restriction site.

For the isolation of a human genomic 1(XI) clone containing exon IIB, a PCR procedure was conducted using total genomic DNA from human leukocytes (Clontech Laboratories, Inc., Palo Alto, CA) with primers RX6 (5`-TGCACCAGAGGATATAATCG-3`) located in exon IIA and HT4 located in exon III (5`-AACATGCCTAGGAGCTTCTG-3`) as described in Ref. 27. A PCR fragment (955 bp) was subcloned into PCRII vector using the TA cloning system (Invitrogen Corp., San Diego, CA) for DNA sequencing. To isolate cDNA for human IIB exon, RT-PCR was performed on human chondrocyte RNA with primers LA1 (5`-AGGAACCTCAGATAGATAG-3`) located in exon I, and VC5 (5`-AAATCATCAACGATGTTTGC-3`) located in exon III (27) . Two PCR products were obtained which after subcloning into the PCRII vector and DNA sequencing were shown to contain exon IIA or exon IIB.

Generation of Specific Probes for Exon IIA or Exon IIB

PCR was performed on plasmid DNAs pXB28 and pXB45 which contain the exon sequences I-IIA-III-IV-V and I-IIB-III-IV-V with sets of primers VS4 (5`-TACACACCAGAAGATTTTAT-3`) and VS5 (5`-CTCTGTTTGTGCTATTGTCT-3`) designed to amplify specifically exon IIA and VC1 (5`-AAAAAGAAAGCCATGGTCAA-3`) and VC2 (5`-CCCTAGTTTGTCTCTTGTTG-3`) designed to amplify specifically exon IIB (see Fig. 6for location of primers). The PCR products were subcloned into PCRII vector and the correct sequences were confirmed.

To obtain biotinylated probes for Northern hybridization, unidirectional PCR was performed using the antisense primer VS5 for IIA and VC2 for IIB and a mixture of dNTPS in which dUTP was biotinylated (Millipore, Bedford, MA). Each template (0.2 mg) was primed with 5 10 M of antisense primer in the presence of 0.5 units of Taq I polymerase (Promega) in reaction buffer with 2.5 m M MgClwith cycle parameters of denaturation (95 °C, 2 min), annealing (54 °C, 1 min), polymerization (72 °C, 3 min) for 40 cycles with a final elongation (75 °C, 5 min). Biotinylated single stranded probes were precipitated with absolute ethanol in the presence of 3 M NaAc, and boiled prior to hybridization.

Northern Hybridization

Total RNA (20 µg/lane) was isolated from 17-day embryonic chicken brain, kidney, gizzard, heart, skeletal muscle, liver, lung, skin, tendon and isolated chondrocytes before being run on a 1% agarose-formamide gel. Transfer was to maximum strength NYTRAN membrane (Schleicher and Schuell, Keene, NH) prehybridized in 5 SSC (1 SCC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5 Denhardt's solution, 0.8% SDS with 100 µg/ml herring sperm DNA (Promega) at 65 °C for 2 h. After washing, membranes were hybridized with single stranded biotinylated exon IIA or exon IIB probes in the same solution as above at 65 °C. Filters were washed twice for 30 min with 2 SSC, 0.1 SDS at 65 °C and once for 15 min in 0.2 SSC, 0.1% SDS at 65 °C. Biotinylated signals were detected with a PolarPlex Chemiluminescent kit (Millipore).

DNA Sequencing

All sequencing was performed on both strands with Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp.). All plasmid templates for sequencing were isolated using the Wizardminipreps DNA purification system (Promega).


RESULTS

Previously, we reported (28) the molecular cloning of human PARP from hyaline cartilage and the subsequent biosynthesis by RT-PCR of a 1.86-kilobase pair product which bridged from PARP to the published nucleotide sequence of the human pro-2(XI) chain (36) . The PCR product was cloned and the complete nucleotide sequence determined. Analysis of this sequence showed that it included a variable region which was found to be much shorter than the variable region of the pro-1(XI) and pro-1(V) chains (Fig. 1). During these experiments, it was found on nucleotide sequencing of individual DNA molecules cloned from the RT-PCR reaction that a short stretch of 63 nucleotides was missing in some clones. The nucleotide-derived amino acid sequence of this stretch was found to be highly acidic (DPTPGEEEEILESSLLPPLEE) and was located in the variable region of the human pro-2(XI) chain (nucleotides 646-708; Ref. 28). This initial observation suggested that differential splicing of the primary transcript may occur in the variable region. Accordingly, RT-PCR reactions were performed with 20-21-mer oligonucleotides which directly span the variable region and using RNA preparations obtained from a variety of human cells in culture. Fig. 2 compares the products of RT-PCR reactions performed for human chondrocytes ( lane 1) and for human skin fibroblasts ( lane 2). In both cases, two major products were observed which were cloned into the PCRII vector using the TA cloning protocol and shown to differ by the expected 63 nucleotides. The same two products were also obtained in varying proportions for human gingival fibroblasts in culture and for a line of human histiocytic lymphoma cells. Small amounts of other products were sometimes observed by RT-PCR (for example, a band of approximately 7-800 nucleotides for human skin fibroblasts; Fig. 2 , lane 2) but were not investigated further. It is possible that additional alternatively spliced forms of the pro-2(XI) chain may also be synthesized during the RT-PCR reaction since two additional exons have recently been identified in transcripts from several mouse tissues but not from mouse cartilage (37, 38) .


Figure 2: RT-PCR reaction performed with primers that span the variable region of the human pro-2(XI) chain. The sense primer (DE1) was located in the PARP domain and the antisense primer (PO2) was located largely in the NC2 domain as described previously (28). Lane 1, human chondrocytes from sternal cartilage. Lane 2, human skin fibroblasts. The location of standard oligonucleotides of different sizes is shown.



Genomic clones coding for the variable region of the pro-2(XI) chain were obtained by screening a human leukocyte library (EMBL 3A) with a 162-bp fragment prepared by PCR using nested primers (LA5 and LA6) on the NZ2 plasmid as a template so that the 63-bp sequence was included. Two overlapping genomic clones were obtained (called HGCol18 and HGCol9) and the whole inserts from each clone were subcloned into plasmid pBluescript II SK+ vector for DNA sequencing. The genomic organization was determined by DNA sequencing reactions performed with primers synthesized from the known cDNA sequence and by PCR reactions using primers which spanned one or more exons. Fig. 3 shows that the 63-bp sequence arises from a single exon which is located between two large introns. In addition, the location of five additional exons was determined for the human pro-2(XI) chain (Fig. 3) and a short stretch of the nucleotide sequence at each intron/exon boundary is shown in Fig. 4. All introns showed canonical sequences for intron/exon junctions.

These results suggested that differential splicing potentially may occur within the variable region of the pro-1(V) and pro-1(XI) collagen chains. Examination of the published sequences for the pro-1(V), pro-1(XI), and pro-2(XI) chains showed the presence of two short stretches of amino acids which span the variable region and are highly conserved for all three chains. One sequence DEEVFEG is located in the ``PARP-like'' domain of chicken pro-1(V) (amino acid positions 210-216; Ref. 22), human pro-1(V) (amino acid positions 213-219; Ref. 25), human pro-1(XI) (amino acid positions 212-218; Ref. 27), and human pro-2(XI) (nucleotide positions 436-456; Ref. 28). A second sequence in the constant region of NC3 (Fig. 1) contains a short sequence, KGQKGEP, for chicken pro-1(V) (positions 448-454; Ref. 22), human pro-1(V) (positions 454-458; Ref. 25), and human pro-1(XI) (positions 425-431; Ref. 27) with one conservative change to KGEKGEP for human pro-2(XI) (28) . This suggested that it may be possible to amplify the variable region of these three chains of the type V/XI family by a single RT-PCR reaction using degenerate oligonucleotides designed from these amino acid sequences. Fig. 5 A shows that numerous bands were obtained when an RT-PCR reaction was performed on 17-day embryonic chicken cartilage RNA using a sense primer 5`-GA(CT)GA(AG)GA(AG)GTITT(CT)GA(AG)GG-3` and an antisense primer 5`-GG(TC)TCICC(TC)TT(TC)T(GC)(TGCA)CC(TC)TT-3` in which some positions were substituted with inosine residues (33, 39) . Each band was cloned into the PCRII vector and the complete nucleotide sequence determined for several of these clones. The results showed that there are at least six different products from the RT-PCR reaction for the chicken pro-1(XI) chain (Fig. 5 B), suggesting a complex pattern of alternative splicing. However, only one PCR product was obtained for the pro-1(V) chain for which the sequence agreed with the previously published nucleotide sequence (22) . For reasons that are unclear, the pro-2(XI) chain has not so far been detected in the products of the RT-PCR reaction. This may be a consequence of designing the degenerate primers by including the sequence of the human pro-2(XI) chain rather than the chicken pro-2(XI) chain for which the sequence is not available. In addition, several products were obtained during the PCR reactions that were found on nucleotide sequencing to arise from priming at additional locations ( e.g. the band of low mobility in Fig. 5 A) and were not investigated further.


Figure 5: A, agarose gel electrophoresis showing the separation of the products of an RT-PCR reaction performed with RNA from two different preparations of chick chondrocytes using degenerate nucleotides which span the variable region of the pro-1(V), pro-1(XI), and pro-2(XI) chains. Each band is designated by its molecular weight and was identified after cloning into the PCRII vector and nucleic acid sequencing (band 735 = pro-1(V); all other bands were found to be derived from the pro-1(XI) chain; 683 = I-IIB-III-IV-V; 653 = I-IIA-III-IV-V; 536 = I-III-IV-V, 437 = I-IIB-III-V; 407 = I-IIA-III-V; 290 = I-III-V. B, diagram showing the organization of six possible variants obtained for the chicken pro-1(XI) chain after RT-PCR performed across the variable region. Note the presence of either IIA or IIB but not both, and the presence or absence of IV.



The six different products of pro-1(XI) from the RT-PCR reaction suggested that a complex pattern of splicing occurs in which there is alternative usage of exon IIA or exon IIB and that exon IV may be present or absent (see below for the genomic structure). A summary of the chicken pro-1(XI) and new human pro-1(XI) sequences is shown in Fig. 6 together with a comparison to the previously published human pro-1(XI) sequence (27) . To examine the intron/exon organization directly a chicken genomic library in the FIX II vector (Stratagene) was screened with fragments generated by a PCR reaction that was performed on two different plasmids with primers PL1 and PL2 located in exons I and V (see Fig. 6) so that both IIA and IIB were included in the probe. Two overlapping genomic clones were obtained and the whole insert of each clone was subcloned from the vector into pBluescript II SK+ for sequencing. The location of the exons was determined from PCR reactions with primers designed for specific exons and is shown in Fig. 7 A. The results showed that the different products observed by RT-PCR arose from alternative splicing of different exons such that either exon IIA or IIB is utilized and exon IV is either present or absent. The sequence of I, IIA, III, IV, and V corresponds to the previously published human cDNA sequence for pro-1(XI) (27) . It was therefore of interest to determine if exon IIB is also present in human genomic DNA. Total human genomic DNA was used as a template for PCR with primers of known sequence located in IIA and III. A PCR product of 955 bp was obtained which was cloned and the presence of exon IIB was demonstrated by DNA sequencing (Fig. 7 B). In addition, it was possible to demonstrate the alternative splicing of exons IIA and IIB by RT-PCR on human chondrocyte RNA with primers located in exon I and exon III so that products were obtained that upon DNA sequencing were found to contain exon IIA or IIB but not both. In Fig. 8 the nucleotide sequences of the intron/exon boundaries of exons IIA, IIB, III, and IV for chicken genomic DNA and exon IIB for human genomic DNA are shown. In addition, the nucleotide and predicted protein sequences of human exon IIB are shown in Fig. 6.

The remarkable feature of these results is the difference in predicted amino acid composition of exon IIB when compared to exons IIA, III, and IV. Exon IIB is highly basic with a calculated pI of 10.6, whereas IIA, III, and IV are all highly acidic with calculated pI values of 3.2, 3.8, and 3.7, respectively. To understand the potential significance of these splicing events, experiments were first performed in which RT-PCR was carried out on 20 µg of total RNA isolated from 17-day chicken embryo tendon, skin, skeletal muscle, lung, liver, kidney, heart, gizzard, cartilage, and brain in which the primers PL1 and PL2 were used which span the variable region (see Fig. 6for location of primers). The results of this experiment are shown in Fig. 9. Some variability can be noted in the products of the RT-PCR reaction in different tissues, but in all tissues transcripts of the pro-1(XI) chain could be detected. However, these experiments involving RT-PCR do not take into consideration differences in level of the initial mRNAs which may be very low in some tissues. Accordingly, Northern analyses were performed to assess the relative amount of expression specifically of exon IIA or IIB with specific biotinylated probes prepared for these exons (Fig. 10). In each lane 20 µg of total RNA was probed. Both exon IIA and exon IIB were easily detected in chicken chondrocytes with two bands of 6.3 and 6.6 kilobases as described previously (8, 9, 27) . In most other tissues, in the conditions of hybridization used, we were unable to detect a signal although our previous RT-PCR analyses demonstrated the presence of low levels of transcripts for the pro-1(XI) chain (Fig. 9). Interestingly, transcripts containing exon II were readily detected in tendon suggesting that there is a high level of transcripts of the pro-1(XI) chain in this tissue. These transcripts were largely of exon IIB.


Figure 9: Agarose gel electrophoresis after RT-PCR performed with RNA (20 µg) from a variety of tissues of 17-day chick embryo using primers PL1 and PL2 (see Fig. 6 for location of primers). Note that all tissues gave a similar profile of products. Lane 1, embryonic chondrocytes; 2, gizzard; 3, heart; 4, kidney; 5, skeletal muscle; 6, tendon.




DISCUSSION

Several interesting examples of differential splicing exist in the connective tissue field (40) . For example, for type II collagen an additional exon is present in the aminopropeptide which is present in precursor cells for cartilage and in noncartilage cells during development, but is apparently absent in mature cartilage (41, 42, 43, 44, 45) . The present analyses show that a region of the pro-1(XI) and pro-2(XI) chains undergoes complex alternative splicing and similar results to ours have recently been reported for the mouse (46) and rat (47) pro-1(XI) chain and the mouse (37, 38) pro-2(XI) chain. It appears that either acidic stretches of sequence are present or absent or, in the case of the pro-1(XI) chain, an acidic stretch is replaced by a very basic stretch of amino acids. The physiological significance of these events is at present unknown but these splicing events are likely to be important since they are conserved between chicken and rat (47) . We have evidence that in the embryonic tendon the basic exon IIB is preferentially utilized (Fig. 10). In addition, this same basic exon is preferentially utilized in the mRNA from the transplantable rat chondrosarcoma (47) . This would suggest that the preferential expression of this exon may be important in establishing the organization of the extracellular matrix in some tissues.


Figure 10: Northern blot prepared with probes specific for exon IIA ( Panel A) or exon IIB ( Panel B) with RNA preparations from a variety of tissues of 17-day chick embryos. Lane 1, tendon; 2, skin; 3, skeletal muscle; 4, lung; 5, liver; 6, kidney; 7, heart; 8, gizzard; 9, chondrocytes (preparation 1); 10, chondrocytes (preparation 2); 11, brain. Note that both IIA and IIB can be detected in chondrocyte preparations but only IIB can be detected in tendon. In the conditions of Northern hybridization, transcripts for IIA or IIB could not be detected in other tissues.



Previously, it was reported that some of the pro-1(V) chains in chicken tendon, but not other tissues, are different and were designated pro-1`(V) (48) . It now appears from the results described in Fig. 10that the pro-1`(V) chain is likely to be the pro-1(XI) chain, and this would agree with other observations that the pro-1(XI) chain has a widespread distribution in tissues (9) . Although the presence of transcripts for the pro-1(XI) chain have not been described previously in tendon, recent results show considerable expression of transcripts for the 1(II) chain in the compressed region of the adult bovine tendon (49) .

Analyses of the biosynthesis of the pro-1(XI) and pro-2(XI) chains (50, 51) , as well as the pro-1(V) chain (52) , all indicate that extensive lengths of each amino-terminal peptide are retained even when these chains are incorporated into fibrils. It would appear that the short collagenous domain called COL2 (Fig. 1) is always retained and in a recent model for type V collagen this domain is proposed to project out from the surface of the fibril (22) . This appears to occur despite the presence of a potential aminopropeptide cleavage site in the NC2 domain between COL2 and COL1 for the pro-1(XI) and pro-1(V) chains (24, 25, 27) . Thus, at least part of the noncollagenous sequences may be located on the surface of the fibrils. For the pro-2(XI) chain there must be cleavage prior to the variable region to give rise to the disulfide bonded fragment called PARP (29) . If this is the only cleavage site for the pro-2(XI) chain then the variable region would be retained on the surface of the fibrils. It is not known if similar PARP-like fragments are formed from the pro-1(XI) and pro-1(V) chains, although these sequences are homologous including the location of the cysteine bridges (30, 31) . However, a peptide antibody prepared to an upstream sequence of the pro-1(XI) chain between the PARP-like domain and the variably spliced region was found by electron microscopy to recognize the thin fibrils, but not the thicker fibrils, of the epiphyseal cartilage of fetal calf and newborn human (53) . Thus, the variable region of pro-1(XI) and pro-2(XI) may be located on the surface of fibrils. In contrast, the NH-terminal sequence was recently determined for the tissue form of the pro-1(V) chain (54) . It was found to be located downstream of comparable regions where differential splicing occurs in the variable region of the pro-1(XI) and pro-2(XI) chains. This agrees with another study which shows using peptide-derived polyclonal antibodies that there is likely to be extensive processing of the tissue form of the pro-1(V) chain but not of the pro-2(V) chain (48) . Thus, the pro-1(V) chain appears to be more extensively processed than the pro-1(XI) and pro-2(XI) chains so that the variable region is largely missing from the fully processed form of the molecule.

The potential presence of a basic sequence (exon IIB of the 1(XI) chain) on the surface of the collagen fibrils of cartilage and other tissues may be important for the interaction of collagen fibrils with the glycosaminoglycan chains of proteoglycans or with hyaluronan. We speculate that such interactions may be of considerable importance in establishing the three-dimensional organization of the matrix of cartilage and the vitreous humor. However, it is also possible that the presence of chains of the type V/XI family of collagens close to the fibril surface may play a role in determining the diameter of each fibril by preventing the addition of further molecules to its surface.


FOOTNOTES

*
This work was supported by Grants AR30481, DE08228, and EY09908 from the National Institutes of Health. The human tissues utilized in this study were obtained through the UAB Cooperative Human Tissue Network which is funded by the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank with accession numbers L38955 for chicken pro-1(XI) and L38956 for human pro-1(XI), exon IIB.

§
To whom correspondence should be addressed: Dept. of Cell Biology, University of Alabama at Birmingham, VH 302, Room 605, Birmingham, AL 35294. Tel.: 205-934-2053; Fax: 205-934-7029; E-mail: rmayne@cellbio.bhs.uab.edu.

The abbreviations are: PARP, proline/arginine-rich protein; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pairs; SSC, standard sodium citrate buffer.


ACKNOWLEDGEMENTS

We thank Pauline M. Mayne, Brian Wood, and Jeanne Holloway for their assistance in the preparation of this manuscript.


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