Structural insights into the mechanism of the DEAH-box RNA helicase Prp43.

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 Prp43•ATP-analog•RNA 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 ß-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.

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.

A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity.

The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.

Impact of the crystallization condition on importin-ß conformation.

In eukaryotic cells, the exchange of macromolecules between the nucleus and cytoplasm is highly selective and requires specialized soluble transport factors. Many of them belong to the importin-ß superfamily, the members of which share an overall superhelical structure owing to the tandem arrangement of a specific motif, the HEAT repeat. This structural organization leads to great intrinsic flexibility, which in turn is a prerequisite for the interaction with a variety of proteins and for its transport function. During the passage from the aqueous cytosol into the nucleus, the receptor passes the gated channel of the nuclear pore complex filled with a protein meshwork of unknown organization, which seems to be highly selective owing to the presence of FG-repeats, which are peptides with hydrophobic patches. Here, the structural changes of free importin-ß from a single organism, crystallized in polar (salt) or apolar (PEG) buffer conditions, are reported. This allowed analysis of the structural changes, which are attributable to the surrounding milieu and are not affected by bound interaction partners. The importin-ß structures obtained exhibit significant conformational changes and suggest an influence of the polarity of the environment, resulting in an extended conformation in the PEG condition. The significance of this observation is supported by SAXS experiments and the analysis of other crystal structures of importin-ß deposited in the Protein Data Bank.

Structural analysis of the spliceosomal RNA helicase Prp28 from the thermophilic eukaryote Chaetomium thermophilum.

Prp28 (pre-mRNA-splicing ATP-dependent RNA helicase 28) is a spliceosomal DEAD-box helicase which is involved in two steps of spliceosome assembly. It is required for the formation of commitment complex 2 in an ATP-independent manner as well as for the formation of the pre-catalytic spliceosome, which in contrast is ATP-dependent. During the latter step, Prp28 is crucial for the integration of the U4/U6·U5 tri-snRNP since it displaces the U1 snRNP and allows the U6 snRNP to base-pair with the 5'-splice site. Here, the crystal structure of Prp28 from the thermophilic fungus Chaetomium thermophilum is reported at 3.2 Šresolution and is compared with the available structures of homologues.

The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction.

The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1's G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1's C-terminal-domain (CTD) and Ntr2. Unlike the NTR, Prp43_Ntr1GP disassembles earlier spliceosomal complexes (A, B, B(act)), indicating that Ntr2/Ntr1-CTD prevents NTR from disrupting properly assembled spliceosomes other than the ILS. The U2 snRNP-intron interaction is disrupted in all complexes by Prp43_Ntr1GP, and in the spliceosome contacts U2 proteins and the pre-mRNA, indicating that the U2 snRNP-intron interaction is Prp43's major target.

Structural and functional analysis of the RNA helicase Prp43 from the thermophilic eukaryote Chaetomium thermophilum.

RNA helicases are indispensable for all organisms in each domain of life and have implications in numerous cellular processes. The DEAH-box RNA helicase Prp43 is involved in pre-mRNA splicing as well as rRNA maturation. Here, the crystal structure of Chaetomium thermophilum Prp43 at 2.9 Šresolution is revealed. Furthermore, it is demonstrated that Prp43 from C. thermophilum is capable of functionally replacing its orthologue from Saccharomyces cerevisiae in spliceosomal disassembly assays.