Despite the considerable body of research dedicated to the field of neurodegeneration, the gap in knowledge on the prion protein and its intricate involvement in brain diseases remains substantial. However, in the past decades, many steps forward have been taken toward a better understanding of the molecular mechanisms underlying both the physiological role of the prion protein and the misfolding event converting it into its pathological counterpart, the prion. This review aims to provide an overview of the main findings regarding this protein, highlighting the advantages of many different animal models that share a conserved amino acid sequence and/or structure with the human prion protein. A particular focus will be given to the species Danio rerio, a compelling research organism for the investigation of prion biology, thanks to its conserved orthologs, ease of genetic manipulation, and cost-effectiveness of high-throughput experimentation. We will explore its potential in filling some of the gaps on physiological and pathological aspects of the prion protein, with the aim of directing the future development of therapeutic interventions.

Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5\' and 3\' splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.

Splicing of mRNA precursors is essential in the regulation of gene expression. U2AF65 recognizes the poly-pyrimidine tract and helps in the recognition of the branch point. Inactivation of fission yeast U2AF65 (Prp2) blocks splicing of most, but not all, pre-mRNAs, for reasons that are not understood. Here, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis in the presence or absence of functional Prp2. Our data indicate that in addition to the splicing elements at the 3\' end of any intron, the nucleotides immediately upstream the intron will determine whether Prp2 is required or dispensable for splicing. By changing those nucleotides in any given intron, we regulate its Prp2 dependency. Our results suggest a model in which Prp2 is required for the coordinated recognition of both intronic ends, placing Prp2 as a key regulatory element in the determination of the exon-intron boundaries.

Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF3- and a poly-U12 RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF3-- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3\' stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

The spliceosome consists of five small RNAs and more than 100 proteins. Almost 50% of the human spliceosomal proteins were predicted to be intrinsically disordered or to contain disordered regions, among them the G-patch protein Spp2. The G-patch region of Spp2 binds to the DEAH-box ATPase Prp2, and both proteins together are essential for promoting the transition from the Bact to the catalytically active B* spliceosome. Here we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that Spp2 is intrinsically disordered in solution. Crystal structures of a complex consisting of Prp2-ADP and the G-patch domain of Spp2 demonstrate that the G-patch gains a defined fold when bound to Prp2. While the N-terminal region of the G-patch always folds into an α-helix in five different crystal structures, the C-terminal part is able to adopt two alternative conformations. NMR studies further revealed that the N-terminal part of the Spp2 G-patch, which is the most conserved region in different G-patch proteins, transiently samples helical conformations, possibly facilitating a conformational selection binding mechanism. The structural analysis unveils the role of conserved residues of the G-patch in the dynamic interaction mode of Spp2 with Prp2, which is vital to maintain the binding during the Prp2 domain movements needed for RNA translocation.

The DEAH-box ATPase Prp2 plays a key role in the activation of the spliceosome as it promotes the transition from the Bact to the catalytically active B* spliceosome. Here, four crystal structures of Prp2 are reported: one of the nucleotide-free state and three different structures of the ADP-bound state. The overall conformation of the helicase core, formed by two RecA-like domains, does not differ significantly between the ADP-bound and the nucleotide-free states. However, intrinsic flexibility of Prp2 is observed, varying the position of the C-terminal domains with respect to the RecA domains. Additionally, in one of the structures a unique ADP conformation is found which has not been observed in any other DEAH-box, DEAD-box or NS3/NPH-II helicase.

Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy. Here, we show that the zinc finger domain is essential for Cwc24 function, while the RING finger domain is dispensable. Cwc24 binds to the spliceosome after the Prp19-associated complex and is released upon Prp2 action. Cwc24 is not required for Prp2-mediated remodeling of the spliceosome, but the spliceosome becomes inactive if remodeling occurs before the addition of Cwc24. Cwc24 binds directly to pre-mRNA at the 5\' splice site, spanning the splice junction. In the absence of Cwc24, U5 and U6 modes of interaction with the 5\' splice site are altered, and splicing is very inefficient, with aberrant cleavage at the 5\' splice site. Our data suggest roles for Cwc24 in orchestrating organization of the spliceosome into an active configuration prior to Prp2-mediated spliceosome remodeling and in promoting specific interaction of U5 and U6 with the 5\' splice site for fidelity of 5\' splice site selection.

Pre-mRNA splicing is an essential step in gene expression that removes intron sequences efficiently and accurately to produce a mature mRNA for translation. It is the large and dynamic RNA-protein complex called the spliceosome that catalyzes intron removal. To carry out splicing the spliceosome not only needs to assemble correctly with the pre-mRNA but the spliceosome requires extensive remodelling of its RNA and protein components to execute the 2 steps of intron removal. Spliceosome remodelling is achieved through the action of ATPases that target both RNA and proteins to produce spliceosome conformations competent for each step of spliceosome activation, catalysis and disassembly. An increasing amount of research has pointed to the spliceosome associated NineTeen Complex (NTC) of proteins as targets for the action of a number of the spliceosomal ATPases during spliceosome remodelling. In this point-of-view article we present the latest findings on the changes in the NTC that occur following ATPase action that are required for spliceosome activation, catalysis and disassembly. We proposed that the NTC is one of the main targets of ATPase action during spliceosome remodelling required for pre-mRNA splicing.

Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome. In the absence of Spp2, the B(act) spliceosome efficiently triggers Prp2\'s NTPase activity, but NTP hydrolysis is not coupled to ribonucleoprotein (RNP) rearrangements leading to catalytic activation of the spliceosome. Transformation of the B(act) to the B* spliceosome occurs only when Spp2 is present and is accompanied by dissociation of Prp2 and a reduction in its NTPase activity. In the absence of spliceosomes, Spp2 enhances Prp2\'s RNA-dependent ATPase activity without affecting its RNA affinity. Our data suggest that Spp2 plays a major role in coupling Prp2\'s ATPase activity to remodeling of the spliceosome into a catalytically active machine.

After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATP-dependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNA-dependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA-RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5\' splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.