Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.     

Complementary transcribed T4 RNA. Association with the polysomes.

Pulse labelled bacteriophage T₄ RNA isolated from polysomes from either early or late infected cells were found to contain complementary RNA. The fraction of complementary late RNA was higher in the heaviest late polysomes. The RNA not associated with polysomes appeared to contain insignificant amounts of complementary RNA. The significance of these findings are discussed.     

Physical properties of the complementary T4 RNA

The complementary transcribed T4 RNA after self-annealing and RNAase treatment was isolated by gel chromatography and then used for further studies. From salt-dependent RNAase resistance and melting studies it is evident that this RNA represents a genuine double-stranded structure. The base content of the isolated double-stranded RNA was found to be the same as total T4 mRNA. Sucrose gradient analysis and hydroxyapatite chromatography of T4 RNA, annealed early and late RNA, and of the isolated double-stranded RNA, gave results indicating that the complementary RNA is part of a RNA molecule and further that the size of the complementary regions are independent of the RNA molecules. Partial digestion of pulse-labelled late RNA with phosphodiesterase I prior to annealing with unlabelled early RNA, showed that the complementary regions on the mRNA are not located to the 5'- or 3'-end but randomly distributed along the T4 RNA molecules.     

Complementary RNA in the chicken sarcoma cells and Rous sarcoma virus]

The subcellular localization in chicken Rous sarcoma of nucleotide sequence, complementary to Rous sarcoma virus RNA was examined by RNA/RNA molecular hybridization. The preparations of radioiodinated virion RNA were annealed with RNAs from different fractions (nuclei, mitochondria, free and membrane-bound polyribosomes) isolated from chicken Rous sarcoma. Formation of RNA-ase resistant hybrids between the viral 125I-RNA and RNA from the mitochondria and membrane-bound polyribosomes was revealed. The latter were characterized by a higher relative redundancy of nucleotide sequences complementary to virion RNA than that in the former, by factor 446. The role of complementary ribonucleotide sequences is discussed.     

Analysis of ribonucleoproteins of influenza virus containing genomic and complementary RNA

The density and sedimentation characteristics of ribonucleoproteins (RNP) containing genomic RNA from influenza virus and RNA complementary have been studied. Radioactive RNA from infected cells has been used for analysis. RNA classes of interest were isolated by reannealing with abundant nonradioactive genomic and complementary RNA and separation of resulting duplexes in electrophoresis. The RNP containing antigenomic virus-specific RNA are practically identical to "genomic" RNP for their sedimentation and density characteristics. The "plus" RNP is characterized by the stoichiometric mode of RNA protein interaction.     

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes

Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA. We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy. A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers. Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe. The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542). The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531). Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.     

Genetic individuality of intracisternal A-particles of Mus musculus.

The nucleic acid sequence relationship between mouse intracisternal type A-particles and type C and B viruses was examined by reciprocal complementary DNA-RNA hybridization; complementary DNAs prepared from the RNAs of intracisternal A-particles were hybridized with high-molecular-weight RNAs from a variety of murine tumor viruses, and complementary DNAs representing a variety of RNA tumor virus genomes were hybridized with the high-molecular-weight RNAs from A-particles. The criterion for homology between two types of virus was that the heterologous hybridization reaction occurs over the same RNA concentration range as the homologous reacton. The results of these hybridizations indicate that there are no major sequence homologies between the RNA of intracisternal A-particles and the RNA of representative members of type B and C viruses of Mus musculus.     

Replication of double-stranded RNA in particles of Penicillium stoloniferum virus S.

RNA polymerase activity was assayed in different particle classes of Penicillium stoloniferum virus S. RNA polymerase activity was found to be associated with H particles, which contain double-stranded RNA and single-stranded RNA, but not with L particles, which contain only double-stranded RNA and not with M particles, which contain only single-stranded RNA. In H particles the reaction occurred with the formation of one new molecule of double-stranded RNA (or two complementary single strands of RNA) per virus particle and the production of product particles (P particles), which contained two molecules of double-stranded RNA (or its equivalent). This RNA polymerase is therefore a replicase, which catalyses the synthesis of the two complementary strands of double-stranded RNA in a single virus particle. This is the first report of this type of RNA polymerase system.