Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo.
U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa 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 to 85% when psoralen crosslinking of nuclei was omitted, or if 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 molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.
D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins / Q000276:immunology / Q000378:metabolism*; D014307:Trioxsalen / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007074:Immunoglobulin G; D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D012313:RNA; C018822:aminomethyltrioxsalen; D014307:Trioxsalen