Nuclear ribonucleoprotein particles probed in living cells.
Contacts between heterogeneous nuclear RNA (hnRNA) and protein in nuclear ribonucleoprotein particles have been photochemically crosslinked in intact HeLa or Friend erythroleukemia cell by irradiation with 254-nm light at doses of 10(1) to 10(5) ergs/mm2 (1 to 10(4) microJ/mm2). The resulting crosslinked particles were isolated and compared with conventional hnRNA . protein (hnRNP) preparations. By the criteria of nuclear fractionation behavior, sedimentation coefficients, nuclease digestion profiles, and RNA-to-protein ratio measured by banding in Cs2SO4 density gradients, the hnRNP particles crosslinked in vivo are identical to nonirradiated particles. Gel blot hybridization of RNA from Friend cell hnRNP crosslinked in vivo reveals that beta-globin RNA sequences remain both intact and hybridizable after the irradiation procedure. The crosslinked hnRNA--protein bonds are stable in 8 M urea/0.5% sodium dodecyl sulfate and withstand centrifugation in Cs2SO4 gradients of initial density 1.50 g/cm3. These results establish that hnRNA is tightly complexed with nuclear proteins in vivo and that hnRNP particles isolated by nuclear fractionation represent native structures.
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D005914:Globins / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D051379:Mice; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000235:genetics; D010777:Photochemistry; D011487:Protein Conformation; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009698:Nucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D005914:Globins; D012260:Ribonucleases