Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles.

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Abstract
The structures and functions of small nuclear ribonucleoprotein (RNP) particles are of interest because of their suggested role in processing heterogeneous nuclear RNA (hnRNA). To determine the conformation of U-1 RNA in U-1 RNP particles and whether proteins of these particles protect segments of U-1 RNA, intact particles and isolated U-1 RNA [from rat Novikoff hepatoma ascites cells] were digested with T1 RNase. The digested particles were immunoprecipitated with anti-Sm antibodies [from sera of systemic lupus erythematosus patients]. 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, a 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 RNP 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 H-bonding with hnRNA splice junctions.