Localisation of PRP8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy

Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 11/2008; 15(11):1206-12. DOI: 10.1038/nsmb.1506
Source: PubMed


The U4/U6-U5 tri-small nuclear ribonucleoprotein (snRNP) is a major, evolutionarily highly conserved spliceosome subunit. Unwinding of its U4/U6 snRNA duplex is a central event of spliceosome activation that requires several components of the U5 portion of the tri-snRNP, including the RNA helicase Brr2, Prp8 and the GTPase Snu114. Here we report the EM projection structure of the Saccharomyces cerevisiae tri-snRNP. It shows a modular organization comprising three extruding domains that contact one another in its central portion. We have visualized genetically tagged tri-snRNP proteins by EM and show here that U4/U6 snRNP forms a domain termed the arm. Conversely, a separate head domain adjacent to the arm harbors Brr2, whereas Prp8 and the GTPase Snu114 are located centrally. The head and arm adopt variable relative positions. This molecular organization and dynamics suggest possible scenarios for structural events during catalytic activation.

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Available from: Patrizia Fabrizio, Feb 18, 2015
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    • "These characteristic domains can be easily identified, not only in class averages (Fig 1C) but also in the raw images (Fig 1D). Anti-TAP antibodies induce specific dimerisation of spliceosomes that contain a single TAP-tagged protein As described earlier (Häcker et al, 2008), proteins were located by immuno-labelling of the tri-snRNP particle, locating the antibody "
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    ABSTRACT: The spliceosome assembles on a pre-mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre-catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno-labelled selected proteins and located them by negative-stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.
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    • "Once again some of the topological features of the module are compatible with physical associations between these factors (e.g., PRP8 with PRP31 or U5-116K or PRP31 with C20ORF14). That knockdown of these late-acting factors, which coordinate the final steps of the splicing process (Bottner et al., 2005; Hä cker et al., 2008; Wahl et al., 2009 "
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    Full-text · Article · Jan 2015 · Molecular Cell
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    • "Better known is the S. cerevisiae ortholog of U5-116 kD, the Snu114p. Häcker et al. could show that the Snu114p occupies a central position within the U4/U6-U5 tri-snRNP particle [14]. Fabrizio et al. concluded that the GTP-binding domain of the U5-116 kD protein plays an important role in either the splicing process itself or the recycling of spliceosomal snRNPs [13]. "
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