Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles.
The structures and functions of small nuclear ribonucleoprotein particles have become of interest because of their suggested role in processing heterogeneous nuclear RNA [Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin S. L. & Steitz, J. A. (1980) Nature (London) 283, 220-224]. To determine the conformation of U-1 RNA in U-1 ribonucleoprotein particles and whether proteins of these particles protect segments of U-1 RNA, intact particles and isolated U-1 RNA were digested with T1 RNase. The digested particles were immunoprecipitated with anti-Sm antibodies. A 5'-end fragment containing nucleotides 1-107 and 3'-end fragments containing nucleotides 108-165 and 108-153 were recovered in nearly quantitative yield from digestion of the particles, suggesting that position 107 is the principal cleavage site in them. At the same T1 RNase concentrations, deproteinized U-1 RNA was cleaved into many fragments. At low T1 RNase concentrations, major cleavage site of deproteinized U-1 RNA was at nucleotide 69. Comparison of the cleavage sites of free U-1 RNA and of U-1 RNA in U-1 ribonucleoprotein particles suggested similar secondary structures. The resistance of the 5' end of U-1 RNA to T1 RNase was unexpected inasmuch as this region has been implicated in hydrogen bonding with heterogeneous nuclear RNA splice junctions.
D000818:Animals; D001483:Base Sequence; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000032:analysis; D012313:RNA; D012342:RNA, Small Nuclear; D051381:Rats; D006163:Ribonuclease T1 / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000032:analysis; D013379:Substrate Specificity
D009698:Nucleoproteins; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D012313:RNA; D012260:Ribonucleases; D006163:Ribonuclease T1