ABSTRACT
Secondary structures of annealed, snapped-back and untreated chicken reticulocyte poly(A)-containing nuclear RNA were investigated by electron microscopy. Three shapes of molecules were observed: 'coat'hanger' shaped, loop-bearing and filamentous molecules. The former two shaped molecules consist of a small and large fold-back or loop plus a short hook or tail, corresponding to the poly(A) segment at the apparent middle of the structure. The third shaped molecule, mainly seen after snap-back treatment, possesses a small fold-back structure at one end only. It is suggested that the small and large fold-backs or loops of the molecule represent the 5'- and 3'-terminal hairpin structures, respectively. Two distinct sizes of molecule, small and large, irrespective of molecule shape, were observed.
Subject(s)
Chickens/blood , Poly A/blood , RNA, Heterogeneous Nuclear/blood , RNA/blood , Reticulocytes/analysis , Animals , Cell Nucleus/analysis , Glyoxal , Hot Temperature , Microscopy, Electron , Nucleic Acid Conformation , Nucleic Acid Denaturation , RNA, Messenger , Reticulocytes/cytologyABSTRACT
The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.
Subject(s)
Furocoumarins , Globins/biosynthesis , RNA, Messenger , Trioxsalen , Animals , Base Sequence , Cell Nucleus/metabolism , Ducks , Erythroblasts/metabolism , Furocoumarins/analogs & derivatives , Molecular Weight , Protein Biosynthesis , RNA, Heterogeneous Nuclear/blood , RNA, Messenger/biosynthesis , Transcription, Genetic , Trioxsalen/analogs & derivativesABSTRACT
A procedure is described for the chromatographic analysis of RNA under fully denaturing conditions on cross-linked Sepharose. Chromatography of DNA . RNA hybrids, poly(C) . poly(G) hybrids and complexes of poly(C) . hnRNA on Sepharose CL in pure formamide at 46 degrees C leads to denaturation and strand separation of the hybrid structures. Using this procedure, nuclear RNA from duck immature red blood cells was resolved according to molecular size and assayed for the presence of globin mRNA sequences by hybridization with complementary DNA. Two size classes of putative globin mRNA precursor molecules were detected at an elution position corresponding to 14--18 S and 23--28 S. As judged from chromatographic analysis on poly(U)-Sepharose, about 70% of the 14--18-S globin precursor RNA is polyadenylated while only 11% of the putative 23--28-S precursor RNA has a poly(A) tract. Inhibition of transcription by actinomycin D and pulse-chase experiments indicate a half-life of less than 7.5 min for these precursor RNA species.
