RNA METABOLISM IN NORMAL AND MALIGNANT GROWTH

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Our earlier results suggested that the ultraviolet radiation target size of human U1 small nuclear RNA (snRNA) transcription may be very large, possibly as much as 4-5 kilobases, although U1 RNA is only 165 nucleotides long. Therefore, the possibility that U1 RNA might be synthesized as very long precursors had to be considered. Analyzing human nuclear RNA gel blots, we have detected recently short-lived large transcripts (a heterodisperse population with an average size of several kilobases) which have sequence homology to U1 DNA. Our major specific aim is to find out what are the large transcripts that contain U1-like sequences. This involves determining if their structure is compatible with being U1 RNA precursors (e.g.: do they contain perfect, full-length U1 RNA sequences?). The next goal is to map these large transcripts and characterize their non-U1 regions. This includes: a) mapping the approximate ends of these transcripts on human genome U1 loci by the nuclease S1 protection assay; b) electron microscopy of RNA-DNA hybrids formed between large transcripts enriched for U1-like sequences and cloned DNA fragments of human U1 gene loci; c) determining if these large transcripts contain regions with homology to any abundant sequence of the human genome other than the segments flanking U1 genes; and d) examining the fate of the non-U1 sequences of these large transcripts in the living cell. If the results from the preceding experiments are compatible with these large transcripts being U1 snRNA precursors, it would be interesting to examine: a) if they are capped by structures that contain either 7-monomethylguanosine or N2,N2,7-trimethylguanosine; and b) some metabolic aspects of these large transcripts (e.g.: the kinetics of disappearance of long transcripts bearing U1-like sequences, in comparison to the appearance of newly made U1 snRNA). Another goal is to characterize the relatively abundant small RNAs that we have detected recently, that have sequence homology to the highly conserved region located immediately upstream from human U1 genes. We have shown that the biosynthesis of human U3 and U4 snRNA, in addition to that of U1 and U2 RNA, is very sensitive to UV radiation. The next goal is to find out if long transcripts bearing sequences like those of either U2, U3 or U4 RNA can be detected. Since this project will yield basic information about the expression of the human genome, its major medical connection is to the study of genetic diseases and neoplasias.