Reconstitution of the U1 small nuclear ribonucleoprotein particle.

Patton, Jeffrey R.; Patterson, Ronald J.; Pederson, Thoru

doi:10.1128/mcb.7.11.4030

Cited by 81 publications

(96 citation statements)

References 44 publications

(43 reference statements)

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“…The secondary structure, but not the sequence, of this stem loop is conserved. Interestingly, deletion of the terminal stem of U1 produced a stable RNA in Xenopus oocytes that was not Sm-precipitable (Jarmolowski and Mattaj 1993), whereas deletion of the terminal stem loop in HeLa extracts altered the sedimentation behavior of the resulting U1 particles (Patton et al 1987). Here, we observed that several phosphates within the terminal stem loop of U1 significantly affect RNP stability when changed to phosphorothioates.…”

Section: A Stem-loop Clamp For the Sm Ringmentioning

confidence: 52%

Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

McConnell¹,

Lokken²,

Steitz³

2003

RNA

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Nucleotide analog interference mapping (NAIM) is a powerful method for identifying RNA functional groups involved in protein-RNA interactions. We examined particles assembled on modified U1 small nuclear RNAs (snRNAs) in vitro and detected two categories of interferences. The first class affects the stability of two higher-order complexes and comprises changes in two adenosines, A65 and A70, in the loop region previously identified as the binding site for the U1 small nuclear ribonucleoprotein (snRNP)-specific U1A protein. Addition of an exocyclic amine to position 2 of A65 interferes strongly with protein binding, whereas removal or modification of the exocyclic amine at position 6 makes little difference. Modifications of A70 exhibit the opposite effects: Additions at position 2 are permitted, but modification of the exocyclic amine at position 6 significantly inhibits protein binding. These interactions, critical for U1A-U1 snRNA recognition in the context of in vitro snRNP assembly, are consistent with previous structural studies of the isolated protein with the RNA hairpin containing the U1A binding site. The second category of interferences affects all partially assembled U1-protein complexes by decreasing the stability of Sm core protein associations. Interestingly, most strong interferences occur at phosphates in the terminal stem-loop region of U1, rather than in the Sm binding site. These data argue that interactions with the phosphate backbone of the terminal stem loop are essential for the stable association of Sm core proteins with the U1 snRNA. We suggest that the stem loop of all Sm snRNAs may act as a clamp to hold the ring of Sm proteins in place.

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Section: A Stem-loop Clamp For the Sm Ringmentioning

confidence: 52%

Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

McConnell¹,

Lokken²,

Steitz³

2003

RNA

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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 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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“…Transcription of the human Ul RNA gene clone pHUI, assembly of Ul snRNP in a HeLa SIO fraction, and glycerol gradient purification were carried out as previously described (26,27 …”

Section: Methodsmentioning

confidence: 99%

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

Patton¹,

Habets²,

Venrooij³

et al. 1989

Mol. Cell. Biol.

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The Ul small nuclear ribonucleoprotein particle (Ul snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to Ul RNA, using a Ul snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of Ul RNA in reconstituted Ul snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the Ul snRNP, with the 5' end of Ul RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the Ul RNA rather than assembling in the Ul snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to Ul RNA in the absence of bound 70K and Sm core proteins.The first mRNA splicing cofactor identified was the Ul small nuclear ribonucleoprotein particle (Ul snRNP). The Ul snRNP binds to the 5' splice site (25) and is required for the first step of splicing, cleavage at the 5' end of the intron (4, 14). Since Ul RNA alone has no apparent specificity for the 5' splice site (25), it is likely that the Ul snRNP is the active cofactor in splicing.The Ul snRNP is a complex of at least nine proteins, termed 70K, A, B', B, C, D, E, F, and G, bound to Ul RNA (reference 5 and references cited therein). Several of these proteins make up the so-called Sm core and are also constituents of the U2, U5, and U4/U6 snRNPs, but the 70K, A, and C proteins are associated only with Ul RNA (3, 5. 11, 13, 28, 35). It is therefore very likely that the 70K, A, and C proteins are related to the unique function of the Ul snRNP.Knowing the structure of the Ul snRNP will be important in understanding how this cofactor functions in mRNA splicing. Toward this objective, we have developed a system for the in vitro assembly of Ul snRNP, using SP6-transcribed human Ul RNA added to a HeLa cell S100 fraction (26). The in vitro-assembled U1 snRNP is very similar to native Ul snRNP (26) and has been used to map the binding site of the 70K protein to the first stem-loop of Ul RNA (27).Other investigators, using Xenioplus oocyte S100 fractions, have found that the first stem-loop of Ul RNA is necessary and sufficient for binding of the 70K, A, and C proteins (9, 10).Human autoantibodies and mouse monoclonal antibodies (1, 2, 16, 17) have been used extensively to study the protein composition, assembly, and functions of the snRNPs (4, 20-23, 26-28, 35, 36). In this investigation, we used an antibody-mediated nuclease protection technique, previ-* Corresponding author. ously used to identify the binding site of the 70K protein (27), to show that the site...

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Reconstitution of the U1 small nuclear ribonucleoprotein particle.

Cited by 81 publications

References 44 publications

Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

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

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

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