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Yu YT, Shu MD, Steitz JA: Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. EMBO J 1998, 17:5783-5795. The U2 small nuclear (sn)RNA undergoes extensive post-transcriptional modifications and contains 2′-O-methylated residues and pseudouridines towards the 5′ end, as well as the 5′ trimethylguanosine cap. Various fragments of natural U2 snRNA were produced by oligonucleotide-directed RNase H cleavage and were then joined with unmodified in vitro transcribed RNA in order to produce chimaeric full-length U2 snRNAs. The ability of modified RNAs to assemble into a fully functional U2 small nuclear ribonucleoprotein (snRNP) was assessed. It was found that modifications of the first 27 residues were important for full assembly into functional U2 snRNPs.
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Crystal structure of the spliceosomal U2B′-U2A′ protein complex bound to a fragment of U2 small nuclear RNA
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Price SR, Evans PR, Nagai K: Crystal structure of the spliceosomal U2B′-U2A′ protein complex bound to a fragment of U2 small nuclear RNA. Nature 1998, 394:645-650. This crystal structure determination provides further insight into RNA recognition. It gives an example of two highly homologous proteins, evolved from a common ancestor, that highly discriminate between similar target RNAs. The authors compare the crystal structures of the U2B″-U2A′ complex with the U1A complex, each bound to their cognate RNA hairpin loop. Only a few amino acid substitutions between U2B″ and U1A result in very specific RNA-binding properties. A detailed analysis reveals how complex formation with U2A′ modulates the RNA-binding specificity of U2B″. This interaction allows key amino acid residues to specifically recognise U2 small nuclear (sn)RNA elements that are different from U1 snRNA and explains the observed specificity.
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Extensive interactions of PRP8 protein with the 5′ and 3′ splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA
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Dix I, Russell CS, O'Keefe RT, Newman AJ, Beggs JD: Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA 1998, 4:1675-1686. The yeast U5 small nuclear ribonucleoprotein (snRNP) was reconstituted using U5 small nuclear RNA either uniformly or site-specifically labelled with photoactivatable 4-thiouridine. After UV irradiation, cross-linked proteins were identified using antibodies or tagged proteins, providing important information on the architecture of the U5 snRNP.
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Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A, Mann M: Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet 1998, 20:46-50. A method that allows the rapid characterisation of the protein components of a large protein assembly such as the spliceosome is described. A biotinylated pre-mRNA substrate was used to purify the spliceosome using affinity chromatography. Individual proteins were isolated from a two-dimensional gel and were rapidly characterised by mass spectrometry and an expressed sequence tag (EST) database search.
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Ban N, Freeborn B, Nissen P, Penczek P, Grassucci RA, Sweet R, Frank J, Moore PB, Steitz TA: A 9 Å resolution X-ray crystallographic map of the large ribosomal subunit. Cell 1998, 93:1105-1115. The authors present the results of their crystallographic studies on the large 50S ribosomal subunit of Haloarcula marismortui. A combination of 20 Å resolution electron microscopy image maps and a large, 18 atom tungsten cluster for heavy-atom phasing yields an electron density map at 9 Å resolution. This map shows more details than anything previously reported. Long, continuous features of the map are consistent with stretches of double-stranded helical RNA. Finally, the molecular architecture of the ribosome begins to reveal its secrets.
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