Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells.
The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.
D002500:Centrifugation, Isopycnic; D055598:Chemical Phenomena; D002621:Chemistry; D005559:Formamides; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008970:Molecular Weight; D009698:Nucleoproteins / Q000032:analysis*; D011061:Poly A / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012332:RNA, Heterogeneous Nuclear / Q000032:analysis / Q000378:metabolism*; D012333:RNA, Messenger; D012261:Ribonucleoproteins / Q000032:analysis* / Q000378:metabolism; D012965:Sodium Chloride; D012967:Sodium Dodecyl Sulfate
D005559:Formamides; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009698:Nucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D011061:Poly A; D012967:Sodium Dodecyl Sulfate; D012965:Sodium Chloride; D012313:RNA