The secondary structure of a messenger RNA precursor probed with psoralen is melted in an<i>in vitro</i>splicing reaction

Wollenzien, Paul; Goswami, Prabhat C.; Teare, John; Szeberényi, József; Goldenberg, Carlos J.

doi:10.1093/nar/15.22.9279

Cited by 15 publications

(12 citation statements)

References 37 publications

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“…Another activity, acting slightly later, blocks further annealing of complementary RNAs and may reflect the sequestering of the pre-mRNA within an RNP complex. A third activity which appears to antagonize duplex formation in the presence of ATP probably corresponds to an ATP-dependent unwinding activity reported previously (Konarska et al, 1985;Ruskin and Green, 1985;Solnick, 1985;Wollenzien et al, 1987). It is possible that one or more of these activities represents factors which interact with pre-mRNA molecules in vivo in the absence of antisense RNA and are involved in splicing.…”

Section: Discussionsupporting

confidence: 53%

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Munroe

1988

The EMBO Journal

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Antisense RNAs complementary to human beta‐globin pre‐mRNA or to a chimeric globin/adenovirus E2a pre‐mRNA specifically and efficiently inhibit pre‐mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre‐mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3′ splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP‐independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre‐mRNA is facilitated, and a closed complex in which pre‐mRNA is sequestered at an early stage of spliceosome assembly.

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Section: Discussionsupporting

confidence: 53%

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Munroe

1988

The EMBO Journal

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“…2B). These results indi-BI 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 (17,28). The pre-mRNAs were gel purified and used as substrates in splicing reactions incubated at 30°C for 90 min (17).…”

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confidence: 96%

“…The pre-mRNAs were gel purified and used as substrates in splicing reactions incubated at 30°C for 90 min (17). The nudear extracts were purified from HeLa cells, H9 cells uninfected or H9 infected with HIV-1 as described (17,28). (17,28).…”

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confidence: 99%

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Virus-Specific Splicing Inhibitor in Extracts from Cells Infected with HIV-1

Gutman

Goldenberg

1988

Science

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Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.

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“…In vitro Transcription The plasmid pTZ19R-HBG was linearized with BamH I and transcribed with T7 RNA polymerase as previously described (15). The resulting 495 nucleotide RNA includes the exon 1, intron 1, and most of exon 2 of human beta globin.…”

Section: Methodsmentioning

confidence: 99%

Specificity of site directed psoralen addition to RNA

Teare¹,

Wollenzien²

1989

Nucl Acids Res

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We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments.

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The secondary structure of a messenger RNA precursor probed with psoralen is melted in anin vitrosplicing reaction

Cited by 15 publications

References 37 publications

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Virus-Specific Splicing Inhibitor in Extracts from Cells Infected with HIV-1

Specificity of site directed psoralen addition to RNA

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