Error-Prone Splicing Controlled by the Ubiquitin Relative Hub1.

Accurate pre-mRNA splicing is needed for correct gene expression and relies on faithful splice site recognition. Here, we show that the ubiquitin-like protein Hub1 binds to the DEAD-box helicase Prp5, a key regulator of early spliceosome assembly, and stimulates its ATPase activity thereby enhancing splicing and relaxing fidelity. High Hub1 levels enhance splicing efficiency but also cause missplicing by tolerating suboptimal splice sites and branchpoint sequences. Notably, Prp5 itself is regulated by a Hub1-dependent negative feedback loop. Since Hub1-mediated splicing activation induces cryptic splicing of Prp5, it also represses Prp5 protein levels and thus curbs excessive missplicing. Our findings indicate that Hub1 mediates enhanced, but error-prone splicing, a mechanism that is tightly controlled by a feedback loop of PRP5 cryptic splicing activation.

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

Structure of yeast U6 snRNPs: arrangement of Prp24p and the LSm complex as revealed by electron microscopy.

Protein components of the U6 snRNP (Prp24p and LSm2-8) are thought to act cooperatively in facilitating the annealing of U6 and U4 snRNAs during U4/U6 di-snRNP formation. To learn more about the spatial arrangement of these proteins in S. cerevisiae U6 snRNPs, we investigated the structure of this particle by electron microscopy. U6 snRNPs, purified by affinity chromatography and gradient centrifugation, and then immediately adsorbed to the carbon film support, revealed an open form in which the Prp24 protein and the ring formed by the LSm proteins were visible as two separate morphological domains, while particles stabilized by chemical cross-linking in solution under mild conditions before binding to the carbon film exhibited a compact form, with the two domains in close proximity to one another. In the open form, individual LSm proteins were located by a novel approach employing C-terminal genetic tagging of the LSm proteins with yECitrine. These studies show the Prp24 protein at defined distances from each subunit of the LSm ring, which in turn suggests that the LSm ring is positioned in a consistent manner on the U6 RNA. Furthermore, in agreement with the EM observations, UV cross-linking revealed U6 RNA in contact with the LSm2 protein at the interface between Prp24p and the LSm ring. Further, LSmp-Prp24p interactions may be restricted to the closed form, which appears to represent the solution structure of the U6 snRNP particle.

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs.

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.