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

Volloch, Vladimir; Schweitzer, Bruce; Rits, Sophia

doi:10.1016/0006-291x(91)91756-3

Cited by 15 publications

(7 citation statements)

References 21 publications

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“…In addition, all of the experiments described so far used antisense RNAs complementary to mRNA but not to pre-mRNA. However, it has been proposed that targeting nuclear RNA, which is much less abundant than its counterpart in the cytoplasm, may enhance the efficiency of the antisense approach (33 Dominski and Kole (22). This approach circumvents the problem of nonspecific inhibition of splicing and might be developed as a therapeutic tool.…”

Section: Discussionmentioning

confidence: 99%

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Takeshima¹,

Nishio²,

Sakamoto³

et al. 1995

J. Clin. Invest.

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Section: Discussionmentioning

confidence: 99%

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Takeshima¹,

Nishio²,

Sakamoto³

et al. 1995

J. Clin. Invest.

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“…If so, antisense molecules may, in addition to their possible participation in RNA replication, also play a role in another aspect of metabolism, common for all mRNAs. In this respect, we would like to point out that, as we demonstrated earlier in a model experiment (26), it is feasible that antisense RNA may serve as a splicing template, thus enhancing processing of pre-mRNA.…”

Section: Discussionmentioning

confidence: 99%

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

Volloch

Schweitzer

Rits

1996

Proc. Natl. Acad. Sci. U.S.A.

148

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The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense 8-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sensestrand globin mRNA.Earlier, we reported the detection and partial characterization of antisense globin RNA in murine erythroleukemia (MEL) cells (1). This RNA appeared to be a complement of the corresponding sense globin mRNA. Indeed, antisense globin RNA had an electrophoretic mobility similar to that of globin mRNA, and it hybridized with probes corresponding to the 5'-and 3'-terminal segments of globin mRNA, as well as with probes corresponding to the entire globin mRNA. Experiments with whole cells (1) and with cytoplasts obtained by enucleation of MEL cells (2) indicated that antisense globin RNA, as well as its sense counterpart, is synthesized in the cytoplasm and suggested that it may serve as an intermediate in cytoplasmic globin mRNA synthesis by RNA-dependent RNA polymerase, an activity that has been detected in erythroleukemia cells (1), as well as in normal reticulocytes (3). If such a process indeed occurred physiologically, one would expect (i) to find the antisense globin RNA molecule not only in MEL cells but also in animal erythroid tissues, and (ii) that these molecules are precise complements of their sense counterparts. Accordingly, the present study was undertaken to test for the occurrence of antisense globin RNA in erythroid cells from mouse tissues and to analyze its primary structure, The publication costs of this article were defrayed in part by page charge...

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“…Alternatively, RNA processing and export might also be vulnerable to interference by antisense RNAs (35). It was shown in vitro (35,36) and suggested in vivo (37,38) that antisense molecules complementary to splice junctions could strongly inhibit RNA processing. However, transport of the intronless herpes simplex virus thymidine kinase gene transcript in mouse L cells was shown to be blocked by antisense RNAs present in the nucleus (39).…”

Section: Discussionmentioning

confidence: 99%

Targeted nuclear antisense RNA mimics natural antisense-induced degradation of polyoma virus early RNA.

Zhong

Batt²,

Carmichael³

1994

Proc. Natl. Acad. Sci. U.S.A.

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We describe a general antisense strategy to inhibit target gene expression. The substitution of a cis-acting ribozyme for a polyadenylylation signal in an antisense expression vector results in the nuclear retention of RNAs and the efficient degradation of their targets. We demonstrate the utility of this system in polyoma virus, where early-strand RNA levels are downregulated in the nucleus by antisense late-strand counterparts. We show that mutations destabizing these naturally occurring antisense trscripts lead to increased levels of earlystrand RNAs. Furthermore, expression in trans of nuclear antisense transcripts lowers early-strand RNA levels and quantitatively mimics the natural regulation.Although artificial antisense techniques have been widely used as a means to repress gene expression, there are few reports in eukaryotic systems ofendogenous antisense RNAs that regulate gene expression under natural circumstances (1)(2)(3)(4)(5). In these systems the mechanisms of inhibition in vivo are poorly understood.Cornelissen (6) proposed two independent mechanisms to describe the action ofantisense RNA. In the first mechanism, a reduction of sense RNA levels is achieved in the nucleus through sense-antisense interactions. Nuclear antisense transcripts might block transcription initiation or elongation, RNA processing, or mRNA transport. RNA duplexes might also induce an endogenous RNase activity. In the second mechanism, antisense RNA exerts its effect in the cytoplasm primarily by affecting translation. In contrast to the effects of nuclear antisense RNA, cytoplasmic antisense RNA usually does not reduce mRNA levels (5).Polyoma virus provides a useful model system to study the effects of nuclear antisense RNA. The polyoma genome is a small circular double-stranded DNA molecule whose early and late transcription units proceed in opposite directions from an intergenic regulatory region. Before viral DNA replication, or in the absence of DNA replication, the early genes (encoding the large, middle, and small tumor antigens) are preferentially expressed, and few late-strand messages are detected (7-15). After the onset of viral DNA replication, there is little increase in the level of early-strand RNA, while the concentration of late-strand transcripts increases rapidly, mirroring the increase in viral DNA copy number (8,(13)(14)(15) In this report, we have explored the repression of polyoma early gene expression by nuclear antisense RNA. We show that destabilization of antisense-early transcripts results in a dramatic increase in the steady-state level of early RNAs. Furthermore, using ribozyme technology we have been able to express and accumulate antisense-early RNA molecules specifically in the nucleus. Overexpression of nuclear antisense-early RNA in trans dramatically reduces polyoma virus early RNAs in a dose-dependent manner and quantitatively mimics the natural regulation. MATERIALS AND METHODSPlasmid Construction. Antisense Constructs. aE-RZ contains the polyoma antisense-early region driven by...

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

Cited by 15 publications

References 21 publications

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Antisense globin RNA in mouse erythroid tissues: structure, origin, and possible function.

Targeted nuclear antisense RNA mimics natural antisense-induced degradation of polyoma virus early RNA.

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