Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2.

The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1's G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43's cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1's C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as 'doorkeepers' and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly

Identification of determinants in the protein partners aCBF5 and aNOP10 necessary for the tRNA:Ψ55-synthase and RNA-guided RNA:Ψ-synthase activities

Protein aNOP10 has an essential scaffolding function in H/ACA sRNPs and its interaction with the pseudouridine(Ψ)-synthase aCBF5 is required for the RNA-guided RNA:Ψ-synthase activity. Recently, aCBF5 was shown to catalyze the isomerization of U55 in tRNAs without the help of a guide sRNA. Here we show that the stable anchoring of aCBF5 to tRNAs relies on its PUA domain and the tRNA CCA sequence. Nonetheless, interaction of aNOP10 with aCBF5 can counterbalance the absence of the PUA domain or the CCA sequence and more generally helps the aCBF5 tRNA:Ψ55-synthase activity. Whereas substitution of the aNOP10 residue Y14 by an alanine disturbs this activity, it only impairs mildly the RNA-guided activity. The opposite effect was observed for the aNOP10 variant H31A. Substitution K53A or R202A in aCBF5 impairs both the tRNA:Ψ55-synthase and the RNA-guided RNA:Ψ-synthase activities. Remarkably, the presence of aNOP10 compensates for the negative effect of these substitutions on the tRNA: Ψ55-synthase activity. Substitution of the aCBF5 conserved residue H77 that is expected to extrude the targeted U residue in tRNA strongly affects the efficiency of U55 modification but has no major effect on the RNA-guided activity. This negative effect can also be compensated by the presence of aNOP10

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing

Author response

The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43ATP-analogRNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a ?-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases

An EST-SSR Linkage Map of Raphanus sativus and Comparative Genomics of the Brassicaceae†

Raphanus sativus (2n = 2x = 18) is a widely cultivated member of the family Brassicaceae, for which genomic resources are available only to a limited extent in comparison to many other members of the family. To promote more genetic and genomic studies and to enhance breeding programmes of R. sativus, we have prepared genetic resources such as complementary DNA libraries, expressed sequences tags (ESTs), simple sequence repeat (SSR) markers and a genetic linkage map. A total of 26 606 ESTs have been collected from seedlings, roots, leaves, and flowers, and clustered into 10 381 unigenes. Similarities were observed between the expression patterns of transcripts from R. sativus and those from representative members of the genera Arabidopsis and Brassica, indicating their functional relatedness. The EST sequence data were used to design 3800 SSR markers and consequently 630 polymorphic SSR loci and 213 reported marker loci have been mapped onto nine linkage groups, covering 1129.2 cM with an average distance of 1.3 cM between loci. Comparison of the mapped EST-SSR marker positions in R. sativus with the genome sequence of A. thaliana indicated that the Brassicaceae members have evolved from a common ancestor. It appears that genomic fragments corresponding to those of A. thaliana have been doubled and tripled in R. sativus. The genetic map developed here is expected to provide a standard map for the genetics, genomics, and molecular breeding of R. sativus as well as of related species. The resources are available at http://marker.kazusa.or.jp/Daikon

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Ψ residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Ψ residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Ψ residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3′-guide sequence is more important than that formed with the 5′-guide sequence. Surprisingly, we showed that a sRNA–rRNA interaction with the targeted uridine in a single-stranded 5′-UNN-3′ trinucleotide instead of the canonical 5′-UN-3′ dinucleotide is functional