RIBONUCLEOPROTEIN ORGANIZATION OF EUKARYOTIC RNA .30. EVIDENCE THAT U1 SMALL NUCLEAR-RNA IS A RIBONUCLEOPROTEIN WHEN BASE-PAIRED WITH PRE-MESSENGER RNA INVIVO

U1 small nucelar RNA is though to be involved in mRNA splicing by binding to complementary sequences in pre-mRNA. Intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA was investigated by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa [human cervical carcinoma] cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen, which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80-85% when psoralen crosslinking of nuclei was omitted, or it the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having MW similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human IgG. The subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is probably a ribonucleoprotein. These proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.