Eva Absmeier, Markus C Wahl
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...
2021: Methods in Molecular Biology
Muhammad Bilal Ahmed, Salman Ul Islam, Jong Kyung Sonn, Young Sup Lee
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...
July 31, 2020: Molecules and Cells
Shouhan Wang, Min Wang, Bin Wang, Jiaqi Chen, Xianbin Cheng, Xiaodan Sun
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...
2020: OncoTargets and Therapy
Naama Sebbag-Sznajder, Yehuda Brody, Hodaya Hochberg-Laufer, Yaron Shav-Tal, Joseph Sperling, Ruth Sperling
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...
2020: Frontiers in Genetics
Dimitrije Stanković, Ann-Katrin Claudius, Thomas Schertel, Tina Bresser, Mirka Uhlirova
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...
May 17, 2020: Disease Models & Mechanisms
S E Cheperegin, A V Malysheva, E P Sannikova, I I Gubaidullin, B D Efremov, D G Kozlov
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...
May 9, 2020: Applied Biochemistry and Biotechnology
Andrea Saltalamacchia, Lorenzo Casalino, Jure Borišek, Victor S Batista, Ivan Rivalta, Alessandra Magistrato
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...
April 10, 2020: Journal of the American Chemical Society
Junqiao Jia, Oleg M Ganichkin, Marco Preußner, Eva Absmeier, Claudia Alings, Bernhard Loll, Florian Heyd, Markus C Wahl
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...
March 20, 2020: Nucleic Acids Research
Karen Vester, Karine F Santos, Benno Kuropka, Christoph Weise, Markus C Wahl
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...
December 30, 2019: Journal of Biological Chemistry
Malini R Capoor, Neelangi Mishra, Sachin Kolte, Gaurav Singla, Arun Gogna, Shivaprakash Rudramurthy, Hariprasath Prakash, Arunaloke Chakrabarti
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...
October 28, 2019: Mycopathologia
Cathleen M Green, Zhong Li, Aaron D Smith, Olga Novikova, Valjean R Bacot-Davis, Fengshan Gao, Saiyang Hu, Nilesh K Banavali, Dennis J Thiele, Hongmin Li, Marlene Belfort
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...
October 2019: PLoS Biology
Nan-Ying Wu, Soo-Chen Cheng
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...
August 28, 2019: Nucleic Acids Research
Xia Chen, Xiaojin Luan, Qianwen Zheng, Chen Qiao, Wanyin Chen, Min Wang, Yidan Yan, Bing Xie, Cong Shen, Zeyu He, Jun Zhang, Mingxi Liu, Xing Hu, Hong Li, Bo Zheng, Jie Fang, Jun Yu
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...
July 10, 2019: Scientific Reports
Hans Garcia Garces, Danielle Hamae Yamauchi, Raquel Cordeiro Theodoro, Eduardo Bagagli
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...
February 2020: Mycopathologia
Zhong Li, Bin Fu, Cathleen M Green, Binbin Liu, Jing Zhang, Yuekun Lang, Sudha Chaturvedi, Marlene Belfort, Guojian Liao, Hongmin Li
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...
2019: Emerging Microbes & Infections
Claire M Smathers, Aaron R Robart
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...
June 13, 2019: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms
E L Garside, T A Whelan, M R Stark, S D Rader, N M Fast, A M MacMillan
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...
May 9, 2019: Journal of Molecular Biology
Tamires A Bitencourt, Felipe B Oliveira, Pablo R Sanches, Antonio Rossi, Nilce M Martinez-Rossi
PURPOSE: Trichophyton rubrum is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in T. rubrum grown in different protein sources and exposed to antifungal drugs. METHODOLOGY: Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of prp1, prp31, prp8 and prp4 kinase genes after exposure of T...
March 22, 2019: Journal of Medical Microbiology
David A Brow
Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as pre-messenger RNA splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function...
March 21, 2019: Genetics
Sebastian M Fica, Chris Oubridge, Max E Wilkinson, Andrew J Newman, Kiyoshi Nagai
During exon ligation, the S. cerevisiae spliceosome recognizes the 3'-splice site (3'SS) of precursor mRNA through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3 Å cryoEM structure of a human post-catalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon-ligation, as shown by functional assays...
January 31, 2019: Science
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