Conserved residues that modulate protein trans-splicing of Npu DnaE split intein

2014 ◽  
Vol 461 (2) ◽  
pp. 247-255 ◽  
Author(s):  
Qin Wu ◽  
Zengqiang Gao ◽  
Yong Wei ◽  
Guolin Ma ◽  
Yuchuan Zheng ◽  
...  
Keyword(s):  
Conserved Residues ◽  
Split Intein ◽  
Trans Splicing ◽  
In Vitro Splicing ◽  
Splicing Efficiency ◽  
Trans Structure

The first crystal trans-structure of Npu DnaE intein identified the role of two conserved residues NArg50 and CSer35 in asparagine cyclization. In vitro splicing assays prove that these two residues synergistically enhance the protein trans-splicing efficiency.

Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components.

RNA ◽  
2021 ◽  
pp. rna.078329.120
Author(s):  
Valentin Jacquier ◽  
Manon Prevot ◽  
Thierry Gostan ◽  
Remy Bordonne ◽  
Sofia Benkhelifa-Ziyyat ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Differential Splicing ◽  
Molecular Determinants ◽  
In Vitro Splicing ◽  
Splicing Efficiency ◽  
Smn Protein ◽  
Survival Of Motor Neuron ◽  
Branchpoint Sequence ◽  
First Time

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

scholarly journals An Off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro

2020 ◽  
Vol 21 (11) ◽  
pp. 4011 ◽  
Author(s):  
Anniina Jaakkonen ◽  
Gerrit Volkmann ◽  
Hideo Iwaï
Keyword(s):  
Emerging Infectious Diseases ◽  
Sars Coronavirus ◽  
Protein Ligation ◽  
Binding Domains ◽  
Naturally Occurring ◽  
Split Intein ◽  
Trans Splicing ◽  
Brevibacillus Choshinensis ◽  
Genetic Fusion

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a “lectibody”. Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

scholarly journals Interfering Expression of Chimeric Transcript SEPT7P2-PSPH Promotes Cell Proliferation in Patients with Nasopharyngeal Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jing Wang ◽  
Guo-Feng Xie ◽  
Yuan He ◽  
Ling Deng ◽  
Ya-Kang Long ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Southern China ◽  
Chromosomal Translocation ◽  
Distinct Type ◽  
Chimeric Transcript ◽  
Trans Splicing

Introduction. Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is mostly prevalent in southern China. The development of NPC involves accumulation of multiple genetic changes. Chromosomal translocation is always thought to be accompanied with the fusion chimeric products. To data, the role of the fusion chimeric transcript remains obscure. Materials and Methods. We performed RNA sequencing to detect the fusion genes in ten NPC tissues. Sanger sequencing and quantitative RT-PCR were used to measure the level of the fusion chimeric transcript in NPC tissues and cell lines. The functional experiments such as CCK8 assay, colony formation, and migration/invasion were conducted to analyze the role of this transcript in NPC in vitro. Results. We demonstrated that the chimeric transcript SEPT7P2-PSPH was formed by trans-splicing of adjacent genes in the absence of chromosomal rearrangement and observed in both NPC patients and cell lines in parallel. Low-expression of the SEPT7P2-PSPH chimeric transcript induced the protein expression of PSPH and promoted cell proliferation, metastasis/invasion, and transforming ability in vitro. Conclusions. Our findings indicate that the chimeric transcript SEPT7P2-PSPH is a product of trans-splicing of two adjacent genes and might be a tumor suppressor gene, potentially having the role of anticancer activity.

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

1990 ◽  
Vol 9 (1) ◽  
pp. 241-249 ◽  
Author(s):  
M. Goux-Pelletan ◽  
D. Libri ◽  
Y. d'Aubenton-Carafa ◽  
M. Fiszman ◽  
E. Brody ◽  
...  
Keyword(s):  
Branch Point ◽  
Point Location ◽  
In Vitro Splicing

scholarly journals The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

1989 ◽  
Vol 9 (11) ◽  
pp. 4852-4861 ◽  
Author(s):  
K Chebli ◽  
R Gattoni ◽  
P Schmitt ◽  
G Hildwein ◽  
J Stevenin
Keyword(s):  
Splice Site ◽  
Site Directed Mutagenesis ◽  
Hairpin Structure ◽  
Cis Acting ◽  
Adenovirus E1a ◽  
Sequence Elements ◽  
In Vitro Splicing ◽  
Hairpin Formation

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

scholarly journals Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 5902-5910 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Jesse Quintero ◽  
Eric S. Reid ◽  
Christian Gocke ◽  
Carl C. Baker
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Splicing Factors ◽  
Bovine Papillomavirus ◽  
Splicing Pattern ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

scholarly journals PRP18, a protein required for the second reaction in pre-mRNA splicing.

1990 ◽  
Vol 10 (1) ◽  
pp. 324-332 ◽  
Author(s):  
U Vijayraghavan ◽  
J Abelson
Keyword(s):  
Micrococcal Nuclease ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Exon 1 ◽  
In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

scholarly journals PRP18, a protein required for the second reaction in pre-mRNA splicing

1990 ◽  
Vol 10 (1) ◽  
pp. 324-332 ◽  
Author(s):  
U Vijayraghavan ◽  
J Abelson
Keyword(s):  
Micrococcal Nuclease ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Exon 1 ◽  
In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

scholarly journals Escape of TLR5 Recognition by Leptospira spp: A Rationale for Atypical Endoflagella

2020 ◽  
Author(s):  
Marion Holzapfel ◽  
Delphine Bonhomme ◽  
Julie Cagliero ◽  
Frédérique Vernel-Pauillac ◽  
Martine Fanton d’Andon ◽  
...  
Keyword(s):  
Innate Immune ◽  
Proteinase K ◽  
Innate Immune Responses ◽  
Conserved Residues ◽  
Proteinase K Treatment ◽  
Deficient Mice ◽  
Wall Components

AbstractLeptospira interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They possess two periplasmic endoflagella that allow their motility. L. interrogans are stealth pathogens that escape the innate immune responses of the NOD-like receptors NOD1/2, and the human Toll-like receptor (TLR)4, sensing peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another receptor of bacterial cell wall components, recognizing flagellin subunits.To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of bioluminescent bacteria or by q-PCR. We did not identify any protective or inflammatory role of murine TLR5 to control pathogenic Leptospira. Likewise, subsequent in vitro experiments showed that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5. However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, although barely mouse TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, using antimicrobial peptides to destabilize live leptospires, we detected some TLR5 activity, suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using different Leptospira strains with mutations in flagellin proteins, we further showed that neither FlaAs nor Fcps participated in the recognition by TLR5, suggesting a role for the FlaBs. These have structural homology to Salmonella FliC, and conserved residues important for TLR5 activation, as shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and in mice downregulated the expression of the FlaBs but not the FlaAs subunits.Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 recognition, our study suggests that the peculiar central localization and stability of the FlaB monomers in the periplasmic endoflagella, associated with the downregulation of FlaB subunits in hosts, constitute an efficient strategy of leptospires to escape TLR5 recognition and the immune response.

scholarly journals Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

2013 ◽  
Vol 79 (9) ◽  
pp. 2968-2978 ◽  
Author(s):  
Aamir Ghafoor ◽  
Zoe Jordens ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glucose Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
Content Type ◽  
Functional Roles ◽  
Polysaccharide Biosynthesis

ABSTRACTPseudomonas aeruginosaproduces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes,pelAtopelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenicpelFdeletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally,in vitroexperiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX7E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ± 3.15% (mean ± standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

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