Type III collagen is found in most connective tissues, but is not found in hyaline cartilage. However, a unique, previously undescribed RNA species has been detected in chick cartilage. This type III collagen- related RNA has an apparent size of 3800 nucleotides, thus is considerably smaller than the authentic 5700 nucleotide type III collagen mRNA. The 3' half of this unique RNA is similar or identical to the authentic type III collagen mRNA. However, exons 1 and 2, as well as additional exons, appear to be missing from the 5' portion of the RNA. This difference in RNA structure suggests that there may be an alternative transcription start site in the type III collagen gene which is preferentially used in cartilage. Thus the absence of type III collagen in cartilage is not due to transcriptional repression of the type III collagen gene or to instability of the type III collagen transcripts in this tissue. Rather, it must be due to some other mechanism, presumably related to the observed alteration in RNA structure. In this application experiments are proposed to determine the structure of the type III collagen-related RNA in cartilage and the role of this RNA in preventing type III collagen production in cartilage. The structure of the cartilage RNA will be determined and compared with the authentic type III collagen mRNA. The 5' half of the chick type III collagen gene will also be isolated, and the promoter(s) and any previously undescribed exons will be identified by comparison of the exon/intron organization with the cDNA sequence. These experiments will indicate whether the structural differences in the cartilage RNA are due to use of an alternative promoter, alternative RNA processing events, or both. They will also define any open reading frames in the RNA, thus indicating whether the RNA is likely to encode a protein (either a non- collagen protein or an altered form of type III collagen). The subcellular distribution of the cartilage type III collagen-related RNA will be determined; these experiments will indicate whether the type III collagen-related RNA is localized primarily in the nucleus or in the cytoplasm, and, if it is found in the cytoplasm, whether it is associated with ribosomes or with ribonucleoprotein particles. These experiments, in conjunction with the sequence data described above, will suggest possible mechanisms preventing type III collagen production in cartilage. If, based on sequence and subcellular distribution data, the type II collagen-related RNA appears to encode a protein, the protein will be isolated and characterized. If the RNA seems unlikely to encode a protein, other possible functions of RNA will be examined. Finally, the cis-acting sequences and trans-acting factors that are important for the function of the promoter(s) of the type III collagen gene in the appropriate tissues will be identified. These experiments will provide increased understanding of the molecular mechanisms that prevent production of type III collagen in cartilage. Furthermore, they will provide more extensive analysis of the structure of the type III collagen gene and its regulation than has heretofore been available.