cDNA cloning and characterization of nucleus confined hnRNA in HeLa cell

Abstract

To understand the structure and physiological function of the hnRNA, cDNA clones for the most typical and abundant hnRNA have been isolated. Pure form of hnRNA was isolate from ribosomal RNA and tRNA by immunoaffinity chromatography with the monoclonal anibody (4F4) to the hnRNP proteins. First strand cDNA was synthesied using random hexamer as a primer and a cDNA library was constructed in $\lambda$ gtll vector. Recombinant phage clones of the hnRNA were isolated from the cDNA librrary by plaque hybridization with $^{32}P$ labeled first strand cDNA of the hnRNA as a probe. Strongly positive plaques were labeled as S series and weakly positive plaques were labeled as W series. It was assumed that the signal intensity and the frequency of the positive plaques were propotional to the abundance of the specific hnRNA. Most of nucleus confined hnRNA contains Alu repeated sequence on the basis of the Southern blot hybridization and nucleotide sequence analysis. Comparison of the alu related sequences the base substitutions of Alu repeated sequences are fairl random. However, 17 bp direct repeats (TTGCAGTGAGCCAAGAT) are well conserved. Another direct repeats was also present in their franking regions. It suggest that Alu repeated sequence are the result of insertion mechanism. The cDNA clone, W16W, hybridized to the discrete hnRNA transcripts in nonpolyadenylated nuclear RNA fraction, but barely to the polyadenylated nuclear RNA and ctoplasmic mRNA, if any. It suggest that the transcripts corresponding to W16W are confined in the nucleus and not polyadenylated in its 3``-termini. It also suggest the expression of the transcript could be controlled at post transcriptional or transport level rather than at transcriptional or translational level. The sie of corresponding hnRNAs are estimated to be about 1.2-1.3 kb. The transcription of the transcript was senstive to Actinomycin D treatment arguing for RNA polymerse II transcript. The transcript of W16W however remains re...