Prp8

Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd...

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans ( Cne ) and Cryptococcus gattii ( Cga ), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay...

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter...

Functional aspects of nucleic acid helicases can be interrogated by various in vitro methods, using purified components, including nucleic acid binding and unwinding assays. Here we describe detailed protocols for the production and purification of the spliceosomal Ski2-like RNA helicase, Brr2, and one of its regulatory factors, the Jab1 domain of the Prp8 protein from yeast. Furthermore, we include a production protocol for radioactively labeled yeast U4/U6 di-snRNA substrate. We describe polyacrylamide gel-based assays to investigate Brr2's RNA binding and unwinding activities...

We have investigated the involvement of the pre-mRNA processing factor 4B (PRP4) kinase domain in mediating drug resistance. HCT116 cells were treated with curcumin, and apoptosis was assessed based on flow cytometry and the generation of reactive oxygen species (ROS). Cells were then transfected with PRP4 or pre-mRNA-processing-splicing factor 8 (PRP8), and drug resistance was analyzed both in vitro and in vivo . Furthermore, we deleted the kinase domain in PRP4 using GatewayTM technology. Curcumin induced cell death through the production of ROS and decreased the activation of survival signals, but PRP4 overexpression reversed the curcumin-induced oxidative stress and apoptosis...

Background: The specific function of pre-mRNA processing factors (Prps) in human malignancies has not been yet investigated. The aim of the present study was to determine the impacts of Prp8 in a common human malignancy, hepatocellular carcinoma (HCC). Materials and Methods: RT-qPCR and Western blotting were performed to measure the expression levels of Prp8 in various HCC cell lines and HCC tissues. A hepatic astrocyte line was transfected with a eukaryotic expression plasmid to overexpress Prp8...

Splicing and alternative splicing of pre-mRNA are key sources in the formation of diversity in the human proteome. These processes have a central role in the regulation of the gene expression pathway. Yet, how spliceosomes are assembled on a multi-intronic pre-mRNA is at present not well understood. To study the spliceosomes assembled in vivo on transcripts with variable number of introns, we examined a series of three related transcripts derived from the β-globin gene, where two transcript types contained increasing number of introns, while one had only an exon...

Retinitis pigmentosa (RP) represents genetically heterogeneous and clinically variable disease characterized by progressive degeneration of photoreceptors resulting in a gradual loss of vision. The autosomal dominant RP type 13 (RP13) has been linked to the malfunction of PRPF8, the essential component of the spliceosome. Over twenty different RP-associated PRPF8 mutations have been identified in human patients. However, the cellular and molecular consequences of their expression in vivo in specific tissue contexts remain largely unknown...

Phospholipase A2 (PLA2 ) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation...

Intron splicing of a nascent messenger RNA transcript by spliceosome (SPL) is a hallmark of gene regulation in eukaryotes. SPL is a majestic molecular machine composed of an entangled network of proteins and RNAs that meticulously promotes intron splicing through the formation of eight intermediate complexes. Cross-communication among the critical distal proteins of the SPL assembly is pivotal for fast and accurately directing the compositional and conformational readjustments necessary to achieve high splicing fidelity...

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114-Prp8 complex co-purified with endogenous GTP...

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudo-enzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive...

We report here a case of disseminated Emergomyces pasteurianus infection from India in a patient with AIDS. The patient presented with weight loss, dyspnoea and multiple non-tender skin lesions over face, neck and chest over 3 months. The case was diagnosed by microscopy, histopathology of sample and isolation of fungus from skin lesion, breast nodule, bone marrow and sputum. The identification of the isolates was confirmed by sequencing internal transcribed spacer region of rDNA, beta-tubulin, actin and intein PRP8...

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus...

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site...

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila...

Inteins (internal proteins) are mobile genetic elements, inserted in housekeeping proteins, with self-splicing properties. Some of these elements have been recently pointed out as modulators of genetic expression or protein function. Herein, we evaluated, in silico, the distribution and phylogenetic patterns of PRP8 intein among 93 fungal strains of the order Onygenales. PRP8 intein(s) are present in most of the species (45/49), mainly as full-length inteins (containing both the Splicing and the Homing Endonuclease domains), and must have transferred vertically in all lineages, since their phylogeny reflects the group phylogeny...

The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essential and non-redundant cellular functions, a Prp8 intein inhibitor is a mechanistically novel antifungal agent. In this report, we demonstrated that cisplatin, an FDA-approved cancer drug, significantly arrested growth of Prp8 intein-containing fungi C. neoformans and C. gattii, but only poorly inhibited growth of intein-free Candida species...

Spliceosomal introns and self-splicing group II introns share a common mechanism of intron splicing where two sequential transesterification reactions remove intron lariats and ligate exons. The recent revolution in cryo-electron microscopy (cryo-EM) has allowed visualization of the spliceosome's ribozyme core. Comparison of these cryo-EM structures to recent group II intron crystal structures presents an opportunity to draw parallels between the RNA active site, substrate positioning, and product formation in these two model systems of intron splicing...

Conformational rearrangements are critical to regulating the assembly and activity of the spliceosome. The spliceosomal protein Prp8 undergoes multiple conformational changes during the course of spliceosome assembly, activation, and catalytic activity. Most of these rearrangements of Prp8 involve the disposition of the C-terminal Jab-MPN and RH domains with respect to the core of Prp8. Here we use X-ray structural analysis to show that a previously characterized and highly conserved β-hairpin structure in the RH domain, that acts as a toggle in the spliceosome, is absent in Prp8 from the reduced spliceosome of the red alga C...