Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Crystal structure of Prp16 in complex with ADP.

DEAH-box helicases play a crucial role in pre-mRNA splicing as they are responsible for major rearrangements of the spliceosome and are involved in various quality-ensuring steps. Prp16 is the driving force during spliceosomal catalysis, remodeling the C state into the C* state. Here, the first crystal structure of Prp16 from Chaetomium thermophilum in complex with ADP is reported at 1.9 Šresolution. Comparison with the other spliceosomal DEAH-box helicases Prp2, Prp22 and Prp43 reveals an overall identical domain architecture. The ß-hairpin, which is a structural element of the RecA2 domain, exhibits a unique position, punctuating its flexibility. Analysis of cryo-EM models of spliceosomal complexes containing Prp16 reveals that these models show Prp16 in its nucleotide-free state, rendering the model presented here the first structure of Prp16 in complex with a nucleotide.

The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR-Cas9 screen.

Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR-Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP-dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target.

Rearrangements within the U6 snRNA Core during the Transition between the Two Catalytic Steps of Splicing.

The RNA catalytic core of spliceosomes as visualized by cryoelectron microscopy (cryo-EM) remains unchanged at different stages of splicing. However, we demonstrate that mutations within the core of yeast U6 snRNA modulate conformational changes between the two catalytic steps. We propose that the intramolecular stem-loop (ISL) of U6 exists in two competing states, changing between a default, non-catalytic conformation and a transient, catalytic conformation. Whereas stable interactions in the catalytic triplex promote catalysis and their disruptions favor exit from the catalytic conformation, destabilization of the lower ISL stem promotes catalysis and its stabilization supports exit from the catalytic conformation. Thus, in addition to the catalytic triplex, U6-ISL acts as an important dynamic component of the catalytic center. The relative flexibility of the lower U6-ISL stem is conserved across eukaryotes. Similar features are found in U6atac and domain V of group II introns, arguing for the generality of the proposed mechanism.

The histone variant H2A.Z promotes splicing of weak introns.

Multiple lines of evidence implicate chromatin in the regulation of premessenger RNA (pre-mRNA) splicing. However, the influence of chromatin factors on cotranscriptional splice site usage remains unclear. Here we investigated the function of the highly conserved histone variant H2A.Z in pre-mRNA splicing using the intron-rich model yeast Schizosaccharomyces pombe Using epistatic miniarray profiles (EMAPs) to survey the genetic interaction landscape of the Swr1 nucleosome remodeling complex, which deposits H2A.Z, we uncovered evidence for functional interactions with components of the spliceosome. In support of these genetic connections, splicing-specific microarrays show that H2A.Z and the Swr1 ATPase are required during temperature stress for the efficient splicing of a subset of introns. Notably, affected introns are enriched for H2A.Z occupancy and more likely to contain nonconsensus splice sites. To test the significance of the latter correlation, we mutated the splice sites in an affected intron to consensus and found that this suppressed the requirement for H2A.Z in splicing of that intron. These data suggest that H2A.Z occupancy promotes cotranscriptional splicing of suboptimal introns that may otherwise be discarded via proofreading ATPases. Consistent with this model, we show that overexpression of splicing ATPase Prp16 suppresses both the growth and splicing defects seen in the absence of H2A.Z.

The Arabidopsis ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development.

In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 facilitates pre-mRNA splicing. However, in Chlamydomonas reinhardtii and Caenorhabditis elegans, Prp16 orthologs are not important for general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. The CLUMSY VEIN (CUV) gene, which encodes a unique Prp16 ortholog in Arabidopsis thaliana, influences auxin-mediated development. A loss-of-function cuv-1 mutation tells us that CUV does not facilitate splicing of pre-mRNA substrates indiscriminately, but differentially effects splicing and expression of genes. Here we show that CUV influences root-meristem maintenance and planar polarity of root-hair positioning, both of which are processes regulated by auxin. We propose that Arabidopsis PRP16/CUV differentially facilitates the expression of genes, including genes involved in auxin biosynthesis, transport, perception and signaling, and that in this way it influences auxin-mediated development.

The NineTeen Complex (NTC) and NTC-associated proteins as targets for spliceosomal ATPase action during pre-mRNA splicing.

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.

Splicing fidelity: DEAD/H-box ATPases as molecular clocks.

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5' splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5' splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5' splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5' splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell.

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.