In vitro reconstitution of 35S ribonucleoprotein complexes.
Ribonucleoprotein complexes (hnRNP) sedimenting at 30-40 S and containing fragments of heterogeneous nuclear RNA (hnRNA) have been extracted from HeLa cell nuclei. Besides hnRNA fragments (8-12 S), the complexes contain eight mostly basic core proteins of Mr 31 000-41 000 as shown by two-dimensional gel electrophoresis. Other proteins (mostly of higher molecular weight) seem to be peripherally associated since they are lost after pelleting and recentrifugation of the hnRNP complexes. The particle dissociates into its protein components after digestion of the endogenous hnRNA fragments by micrococcal nuclease. After inactivation of the nuclease and addition of a wide variety of exogenous RNAs [MS2 phage RNA, poly(U), poly(C), poly(A), and poly(A,U)], a RNP particle is re-formed which resembles the native hnRNP complex according to its sedimentation value (35 S), its appearance in the electron microscope, its density in metrizamide, and its protein composition. No particles are formed on double-stranded RNA [poly(A) . poly(U)] or native DNA whereas denatured DNA allows complex formation. On MS2 RNA (3569 nucleotides), the formation of tri- and tetrameric complexes is observed. This indicates the presence of 900-1200 nucleotides per particle. In vivo, 40S hnRNP particles are a unit component of larger RNP structures. Hence, we conclude from our results that the hnRNP core proteins have the intrinsic capability to associate with nascent single-stranded hnRNA regions to form these RNP complexes. Because of the lack of any sequence specificity, the complexes may function in packaging of the hnRNA and in connection with other nuclear components may provide a scaffold for subsequent processing reactions.
D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008970:Molecular Weight; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012334:RNA, Neoplasm; D012261:Ribonucleoproteins