In vitro reconstitution of snRNPs: a reconstituted U4/U6 snRNP participates in splicing complex formation.

Pikielny, Claudio W.; Bindereif, Albrecht; Green, M R

doi:10.1101/gad.3.4.479

Cited by 42 publications

(35 citation statements)

References 50 publications

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“…There are many examples of reconstitution of snRNPs using X. laevis egg extracts or HeLa cell S100 extracts IHamm et al 1987IHamm et al , 1988Patton et al 1987;Patton and Pederson 1988;Kleinschmidt et al 1989;Pikielny et al 1989) and examples of functional assembly of human snRNA with amphibian or HeLa proteins (Krainer 1988;Pan and Prives 1988). This work and that of McPheeters et al (1989) are the first examples of synthetic U-RNAs that are able to restore splicing in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…In light of the recent finding that h u m a n U1 and U2 snRNAs are capable of forming functional snRNPs with amphibian proteins (Pan and Prives 1988), it was interesting to Functional reconstitution of U6 snRNPs know whether the h u m a n U6 RNA could recover splicing activity in extracts depleted of yeast U6, associate with yeast splicing proteins, and interact with yeast U4. To test this possibility, h u m a n U6 RNA, synthesized in vitro using SP6 RNA polymerase (Pikielny et al 1989), was added to the oligonucleotide-treated extract, and the restoration of splicing activity was monitored by the addition of actin pre-mRNA {Fig. 9A, lanes 6-8).…”

Section: Interesting Differences Between Yeast and Human U6mentioning

confidence: 99%

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In vitro assembly of yeast U6 snRNP: a functional assay.

Fabrizio¹,

McPheeters²,

Abelson³

1989

Genes & Development

130

110

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U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the TTU6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.

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Section: Discussionmentioning

confidence: 99%

Section: Interesting Differences Between Yeast and Human U6mentioning

confidence: 99%

In vitro assembly of yeast U6 snRNP: a functional assay.

Fabrizio¹,

McPheeters²,

Abelson³

1989

Genes & Development

130

110

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“…The existence of stem I has been established by psoralen cross-linking experiments (Rinke et al 1985). In vitro reconstitution studies have shown that both stem I and stem II are required for the formation of the U4/U6 snRNP (Hamm and Mattaj 1989;Pikielny et al 1989;Bindereif et al 1990;Vankan et al 1990). The extensive base-pairing interaction between U4 and U6 is consistent with the observation that the U4/U6 snRNP is very stable in vitro (T m ~53°C} [Brow and Guthrie 1988); this has prompted speculation that an ATP-dependent RNA helicase may mediate the unwinding event.…”

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confidence: 99%

Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs.

Shannon

Guthrie²

1991

Genes Dev.

147

167

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U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing. U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo. Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth. Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites. Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells. The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP. We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.

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“…Several groups have described systems for the in vitro reconstitution of mammalian snRNPs with physical and immunological properties identical to authentic snRNPs (Harem et al 1987(Harem et al , 1989Patton et al 1987;Riedel et al 1987;Patton and Pederson 1988;Kleinschmidt et al 1989;Pikielny et al 1989). However, none of these in vitro assembled snRNPs has been shown to be functional in pre-mRNA splicing.…”

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confidence: 99%

In vitro reconstitution of functional yeast U2 snRNPs.

McPheeters¹,

Fabrizio²,

Abelson³

1989

Genes & Development

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A system for the functional reconstitution of yeast U2 snRNPs using synthetic U2 RNAs is described. We use oligonucleotide-directed RNase H cleavage to specifically deplete yeast extracts of their endogenous full-length U2 snRNA and consequently inactivate pre-mRNA splicing activity. The subsequent addition of synthetic yeast U2 RNAs, derived by in vitro transcription (T7U2 RNAs), to these oligonucleotide-treated extracts efficiently reconstitutes their ability to splice pre-mRNA. The use of deletion derivatives of the T7U2 RNA has demonstrated that the region downstream from the conserved Sm-binding site sequence in the yeast U2 RNA is not absolutely required for pre-mRNA splicing activity in vitro. Furthermore, we found that both human and rat U2 RNAs can function in yeast extracts. We also show that point mutations in the yeast U2 RNA can be analyzed using the in vitro reconstitution system. Allele-specific suppression of mutations in pre-mRNA branch site sequence is observed when the appropriate compensatory mutations in the branch site recognition region of the T7U2 RNA are introduced. Finally, we present a model for the interaction of the U2 and U6 snRNAs during pre-mRNA splicing.

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In vitro reconstitution of snRNPs: a reconstituted U4/U6 snRNP participates in splicing complex formation.

Cited by 42 publications

References 50 publications

In vitro assembly of yeast U6 snRNP: a functional assay.

In vitro assembly of yeast U6 snRNP: a functional assay.

Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs.

In vitro reconstitution of functional yeast U2 snRNPs.

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