Mechanism of spliceosome remodeling by the ATPase/helicase Prp2 and its coactivator Spp2

Science ◽  
2020 ◽  
Vol 371 (6525) ◽  
pp. eabe8863
Author(s):  
Rui Bai ◽  
Ruixue Wan ◽  
Chuangye Yan ◽  
Qi Jia ◽  
Jianlin Lei ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Adenosine Triphosphatase ◽  
Biochemical Analysis ◽  
Mrna Splicing ◽  
Structural Basis ◽  
Atomic Structures

Spliceosome remodeling, executed by conserved adenosine triphosphatase (ATPase)/helicases including Prp2, enables precursor messenger RNA (pre-mRNA) splicing. However, the structural basis for the function of the ATPase/helicases remains poorly understood. Here, we report atomic structures of Prp2 in isolation, Prp2 complexed with its coactivator Spp2, and Prp2-loaded activated spliceosome and the results of structure-guided biochemical analysis. Prp2 weakly associates with the spliceosome and cannot function without Spp2, which stably associates with Prp2 and anchors on the spliceosome, thus tethering Prp2 to the activated spliceosome and allowing Prp2 to function. Pre-mRNA is loaded into a featured channel between the N and C halves of Prp2, where Leu536 from the N half and Arg844 from the C half prevent backward sliding of pre-mRNA toward its 5′-end. Adenosine 5′-triphosphate binding and hydrolysis trigger interdomain movement in Prp2, which drives unidirectional stepwise translocation of pre-mRNA toward its 3′-end. These conserved mechanisms explain the coupling of spliceosome remodeling to pre-mRNA splicing.

Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

scholarly journals Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

2014 ◽  
Vol 70 (6) ◽  
pp. 1622-1630 ◽  
Author(s):  
Sina Möhlmann ◽  
Rebecca Mathew ◽  
Piotr Neumann ◽  
Andreas Schmitt ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Binding Activity ◽  
Mrna Splicing ◽  
Structural Basis ◽  
Atp Binding ◽  
Helicase Domain ◽  
Crucial Step ◽  
Closed Conformation ◽  
Dead Box ◽  
Open Conformation ◽  
U1 Snrnp

The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Structural Basis for Polypyrimidine Tract Recognition by the Essential Pre-mRNA Splicing Factor U2AF65

2006 ◽  
Vol 23 (1) ◽  
pp. 49-59 ◽  
Author(s):  
E. Allen Sickmier ◽  
Katherine E. Frato ◽  
Haihong Shen ◽  
Shanthi R. Paranawithana ◽  
Michael R. Green ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Splicing Factor ◽  
Structural Basis ◽  
Polypyrimidine Tract

scholarly journals OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L

2007 ◽  
Vol 178 (5) ◽  
pp. 749-755 ◽  
Author(s):  
Zhiyin Song ◽  
Hsiuchen Chen ◽  
Maja Fiket ◽  
Christiane Alexander ◽  
David C. Chan
Keyword(s):  
Membrane Potential ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Mrna Splicing ◽  
Mitochondrial Fusion ◽  
Mrna Splice ◽  
Mitochondrial Processing Peptidase ◽  
Processing Peptidase ◽  
Guanosine Triphosphatase ◽  
Splice Forms

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.

Structural basis for messenger RNA movement on the ribosome

Nature ◽  
2006 ◽  
Vol 444 (7117) ◽  
pp. 391-394 ◽  
Author(s):  
Gulnara Yusupova ◽  
Lasse Jenner ◽  
Bernard Rees ◽  
Dino Moras ◽  
Marat Yusupov
Keyword(s):  
Messenger Rna ◽  
Structural Basis

scholarly journals Splice site mutations are a common cause of X-linked chronic granulomatous disease

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1553-1558 ◽  
Author(s):  
M de Boer ◽  
BG Bolscher ◽  
MC Dinauer ◽  
SH Orkin ◽  
CI Smith ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Splice Site ◽  
Messenger Rna ◽  
Mrna Splicing ◽  
Molecular Defect ◽  
Granulomatous Disease ◽  
Splice Sites ◽  
Nucleotide Substitutions ◽  
Common Cause ◽  
Donor Splice

Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). We studied the molecular defect in four patients with X- linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. Gp91-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7, 5, 3, and 2 sequences in patients 1, 2, 3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3′-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide substitutions within the first five positions of the down-stream 5′ donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3′ acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.

scholarly journals Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2

2021 ◽  
Vol 118 (10) ◽  
pp. e2020587118
Author(s):  
Jingjing Pei ◽  
Nicole D. Wagner ◽  
Angela J. Zou ◽  
Srirupa Chatterjee ◽  
Dominika Borek ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Human Respiratory Syncytial Virus ◽  
Host Responses ◽  
Structural Basis ◽  
Downstream Signaling ◽  
Nonstructural Protein 2 ◽  
Syncytial Virus

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I–like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNβ messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

ANGEL2 is a member of the CCR4 family of deadenylases with 2′,3′-cyclic phosphatase activity

Science ◽  
2020 ◽  
Vol 369 (6503) ◽  
pp. 524-530
Author(s):  
Paola H. Pinto ◽  
Alena Kroupova ◽  
Alexander Schleiffer ◽  
Karl Mechtler ◽  
Martin Jinek ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Mrna Splicing ◽  
Rna Molecules ◽  
Unfolded Protein ◽  
Rna Pathways ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Cyclic Phosphates ◽  
Hydrolysis Of

RNA molecules are frequently modified with a terminal 2′,3′-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2′,3′-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2′,3′-cyclic phosphates into 2′,3′-OH nucleotides. We analyzed ANGEL2’s substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box–binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)–mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2′,3′-cyclic phosphates.

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