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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    • "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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    ABSTRACT: Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation
    Molecular Cell 01/2015; DOI:10.1016/j.molcel.2014.10.030 · 14.02 Impact Factor
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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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    ABSTRACT: Mutations in EFTUD2 were proven to cause a very distinct mandibulofacial dysostosis type Guion-Almeida (MFDGA, OMIM #610536). Recently, gross deletions and mutations in EFTUD2 were determined to cause syndromic esophageal atresia (EA), as well. We set forth to find further conditions caused by mutations in the EFTUD2 gene (OMIM *603892).Methods and resultsWe performed exome sequencing in two familial cases with clinical features overlapping with MFDGA and EA, but which were previously assumed to represent distinct entities, a syndrome with esophageal atresia, hypoplasia of zygomatic complex, microcephaly, cup-shaped ears, congenital heart defect, and intellectual disability in a mother and her two children [AJMG 143A(11):1135-1142, 2007] and a supposedly autosomal recessive oto-facial syndrome with midline malformations in two sisters [AJMG 132(4):398-401, 2005].While the analysis of our exome data was in progress, a recent publication made EFTUD2 mutations highly likely in these families. This hypothesis could be confirmed with exome as well as with Sanger sequencing. Also, in three further sporadic patients, clinically overlapping to these two families, de novo mutations within EFTUD2 were identified by Sanger sequencing. Our clinical and molecular workup of the patients discloses a broad phenotypic spectrum, and describes for the first time an instance of germline mosaicism for an EFTUD2 mutation. The clinical features of the eight patients described here further broaden the phenotypic spectrum caused by EFTUD2 mutations or deletions. We here show, that it not only includes mandibulofacial dysostosis type Guion-Almeida, which should be reclassified as an acrofacial dysostosis because of thumb anomalies (present in 12/35 or 34% of patients) and syndromic esophageal atresia [JMG 49(12). 737-746, 2012], but also the two new syndromes, namely oto-facial syndrome with midline malformations published by Megarbane et al. [AJMG 132(4): 398-401, 2005] and the syndrome published by Wieczorek et al. [AJMG 143A(11): 1135-1142, 2007] The finding of mild phenotypic features in the mother of one family that could have been overlooked and the possibility of germline mosaicism in apparently healthy parents in the other family should be taken into account when counseling such families.
    Orphanet Journal of Rare Diseases 07/2013; 8(1):110. DOI:10.1186/1750-1172-8-110 · 3.36 Impact Factor
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    • "Brr2p interaction sites were identified in the central domain, stem I, and 59 SL areas of U4 but not on the U4 side of stem II (Fig. 2C), making it implausible that stem II is a substrate of Brr2p. This in turn suggests that Brr2p may unwind only stem I. Structural analyses of the S. cerevisiae U4/U6:U5 trisnRNP suggested that U4/U6 stem II coaxially stacks on the U4 59 SL, forming a separate domain from stem I (Lescoute and Westhof 2006; Hä cker et al. 2008). Therefore , to investigate the importance of the U4 39 SL for Brr2p interaction with the U4/U6 duplex, we performed electrophoretic gel mobility shift assay (EMSA) experiments using Brr2 protein purified from yeast (Supplemental Fig. S2) with in vitro transcribed RNAs. "
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    ABSTRACT: Brr2p is one of eight RNA helicases involved in pre-mRNA splicing. Detailed understanding of the functions of Brr2p and other spliceosomal helicases has been limited by lack of knowledge of their in vivo substrates. To address this, sites of direct Brr2p-RNA interaction were identified by in vivo UV cross-linking in budding yeast. Cross-links identified in the U4 and U6 small nuclear RNAs (snRNAs) suggest U4/U6 stem I as a Brr2p substrate during spliceosome activation. Further Brr2p cross-links were identified in loop 1 of the U5 snRNA and near splice sites and 3' ends of introns, suggesting the possibility of a previously uncharacterized function for Brr2p in the catalytic center of the spliceosome. Consistent with this, mutant brr2-G858R reduced second-step splicing efficiency and enhanced cross-linking to 3' ends of introns. Furthermore, RNA sequencing indicated preferential inhibition of splicing of introns with structured 3' ends. The Brr2-G858Rp cross-linking pattern in U6 was consistent with an open conformation for the catalytic center of the spliceosome during first-to-second-step transition. We propose a previously unsuspected function for Brr2p in driving conformational rearrangements that lead to competence for the second step of splicing.
    Genes & development 11/2012; 26(21):2408-21. DOI:10.1101/gad.199307.112 · 10.80 Impact Factor
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