Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen.

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Abstract
The psoralen derivative aminomethyltrioxsalen (AMT, 4''-aminomethyl-4,5'',8-trimethylpsoralen) was employed as a probe for heterogeneous nuclear RNA (hnRNA) double-stranded regions in experiments with living HeLa [human cervical carcinoma] cells. hnRNA/ribonucleoprotein (hnRNP) particles were purified from untreated or AMT-treated cells after irradiation with 365-nm light, and double-stranded hnRNA regions (dsRNA) were isolated by RNase A and RNase T1 digestion of hnRNP, followed by preparative Cs2SO4 isopycnic centrifugation. The purified, hnRNP-derived dsRNA was then assayed for interstrand crosslinks by measurement of its snapback to RNase-resistant form after thermal denaturation. By this procedure, the amount of crosslinked dsRNA increased 3- to 7-fold in cells exposed to AMT in vivo. The levels of crosslinking in vivo compared favorably with those observed in model experiments with pure dsRNA in vitro. Double-stranded hnRNA regions apparently exist in the living cell. Base-paired regions are apparently organized as rather accessible sites within the nucleus.