Intermolecular Duplex Research Articles

Interstrand duplexes in friend erythroleukemia nuclear RNA: The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% ( v v ) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4′-aminomethyl-4,5′,8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. β-Globin mRNA but not β-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A) + molecules that were free of interactions with other RNAs. β-Globin pre-mRNA belongs to this class. The second class included poly(A) + molecules that contained interstrand duplexes. β-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA. The fact that most of the hybrids are detected in non-polyadenylated RNA lends support to the concept that they are necessary for RNA processing and that processing precedes polyadenylation. Finally, the fact that small nuclear RNAs not required for splicing form stable hybrids with heterogeneous nuclear RNA is a further indication of the complexity of functions of these RNA-RNA interactions.

Transcription of repeated sequences of the mouse B1 family in friend erythroleukaemic cells: Intermolecular RNA-RNA duplex formation between polyadenylated and non-polyadenylated nuclear RNAs

It has been shown by previous workers that heterogeneous nuclear RNA in eucaryotic cells exists in the form of a complex with small nuclear RNA molecules, which are involved in RNA processing. It is demonstrated in this report that polyadenylated RNAs from the nuclei of Friend erythroleukaemic cells are isolated as complexes with a wide spectrum of non-polyadenylated RNAs. The associated non-polyadenylated RNAs are mainly of high molecular weight (15 to 30 S), but include the 4.5 S and 7 S RNA species. The complexes can be disrupted by denaturation with dimethyl sulphoxide, but also can be reconstituted by reannealing under appropriate stringency conditions. Saturation experiments involving unique DNA probes have shown that the associated non-polyadenylated RNA has a high base sequence complexity. It is shown that complexes involving poly(A) + nuclear RNA contain transcripts of the highly repeated mouse B1 family. The vast majority of these repetitive sequence transcripts are removed by processing within the nucleus and are barely detectable in poly(A) + polysomal RNA. Double-stranded RNA prepared from the nuclear complexes by digestion with ribonucleases contains sequences complementary to the mouse B1 family, suggesting that these may be involved in duplex formation between polyadenylated and non-polyadenylated RNAs. The possible role of interrnolecular RNA-RNA duplexes involving repetitive sequences in the folding of messenger RNA precursors prior to removal of introns and splicing is discussed.

Transcription of complementary repeat sequences in amphibian oocytes

Repeat sequences are transcribed in the germinal vesicles of amphibian oocytes. In the hnRNA population both complements of the repeats are found and can be readily detected because they form intermolecular duplex structures. The structure and formation of duplex regions have been studied in the hnRNA of Xenopus laevis, Triturus cristatus, Amphiuma means and Necturus maculosus, a series of amphibians of increasing genome size (C-value). In T. cristatus, the duplex structures are mostly 600-1200 bp in length, whereas in X. laevis they are shorter and in N. maculosus they tend to be longer. Although the proportion of RNA sequence capable of rapidly forming duplex structures is different in different organisms, this property bears no relationship to C-value. However the sequence complexity of complementary repeats, as estimated from the rate of duplex formation, does show an increasing trend with C-value. The complementary repeats found in oocyte hnRNA are transcribed from families of DNA sequence that are each represented in the genome by thousands of copies. The extent of cross-species hybridization is low, indicating that the repeat sequences transcribed in different amphibian genera are not the same. In situ hybridization experiments indicate that the repeat sequences are spread throughout the genome. The evolution and possible function of complementary repeats are considered.

Structural organization of nascent transcripts and hnRNA molecules in amphibian oocytes.

Comparisons of relative lengths of lampbrush loops, nascent RNP transcripts and hnRNA molecules from oocytes of amphibia with different C-values show that there is an increasing trend in loop, and transcriptional unit, length with increase in genome size but no increasing trend with respect to RNA contour length. The formation of duplex regions and circles in RNP fibrils indicates that RNA processing may occur within the nascent fibrils. The hnRNA molecules from oocytes of the various amphibia readily form intermolecular duplex structures. These complementary sequences have a low kinetic complexity and are transcribed from highly repetitive sequences distributed throughout the genome. Their possible function is considered.

Intermolecular duplexes formed from polyadenylylated vaccinia virus RNA.

Approximately 15% of the polyadenylic acid-containing cytoplasmic RNA labeled from 5 to 7 h after vaccinia virus infection formed intermolecular duplex structures characterized as double-stranded RNA by RNase resistance, density in Cs2SO4, base composition, chromatography on cellulose, and ability to inhibit reticulocyte cell-free protein synthesis. Both sucrose gradient sedimentation and electron microscopic analysis indicated that the double-stranded regions were several hundred to more than a thousand nucleotide base pairs long. The double-stranded RNA, after denaturation, hybridized to approximately 25% of the vaccinia virus genome, whereas total late RNA hybridized to 42%. The finding that the duplex RNA, after denaturation, hybridized to most HindIII restriction endonuclease fragments of vaccinia virus DNA indicated that symmetrical transcription is not confined to the terminal inverted repeat sequence or to one contiguous region of the genome. Although relatively little labeled, early, polyadenylic acid-containing RNA formed RNase-resistant hybrids upon self-annealing, the percentage increased upon addition of unlabeled late RNA, indicating that the latter contains "anti-early" sequences.

Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells

Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal — that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.