scholarly journals psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lisa M. Strittmatter ◽  
Charlotte Capitanchik ◽  
Andrew J. Newman ◽  
Martina Hallegger ◽  
Christine M. Norman ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Splice Site ◽  
Rna Binding ◽  
Mrna Splicing ◽  
Structural Studies ◽  
Rna Helicases ◽  
Exon Ligation ◽  
Before And After ◽  
The Stability

AbstractRNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3′-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

scholarly journals PsiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

2020 ◽  
Author(s):  
Lisa M. Strittmatter ◽  
Charlotte Capitanchik ◽  
Andrew J. Newman ◽  
Martina Hallegger ◽  
Christine M. Norman ◽  
...  
Keyword(s):  
Rna Binding ◽  
Positional Information ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Imaging Studies ◽  
Rna Helicases ◽  
Binding Profile ◽  
Exon Junction ◽  
Exon Ligation ◽  
Before And After

AbstractEight RNA helicases remodel the spliceosome to effect pre-mRNA splicing but their mechanism of action remains poorly understood. We have developed “purified spliceosome iCLIP” (psiCLIP) to define helicase-RNA contacts in specific spliceosomal states. psiCLIP reveals previously unappreciated dynamics of spliceosomal helicases. The binding profile of the helicase Prp16 is influenced by the distance between the branch-point and 3’ splice site, while Prp22 binds diffusely on the intron before exon ligation but switches to more narrow binding downstream of the exon junction after exon ligation. Notably, depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, demonstrating that psiCLIP can be used to study the relationships between helicases and auxiliary splicing factors. Thus, psiCLIP is sensitive to spliceosome dynamics and complements the insights from structural and imaging studies by providing crucial positional information on helicase-RNA contacts during spliceosomal remodeling.

scholarly journals Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

2018 ◽  
Author(s):  
Xiaofeng Zhang ◽  
Xiechao Zhan ◽  
Chuangye Yan ◽  
Wenyu Zhang ◽  
Dongliang Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Splice Site ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Exon Ligation ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Nuclear Rna ◽  
Before And After ◽  
Functional States

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

scholarly journals Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.

1995 ◽  
Vol 15 (11) ◽  
pp. 6273-6282 ◽  
Author(s):  
X Peng ◽  
S M Mount
Keyword(s):  
Splice Site ◽  
Rna Processing ◽  
Rna Binding ◽  
Critical Role ◽  
Mrna Splicing ◽  
Genetic Enhancement ◽  
X Rays ◽  
Loss Of Function ◽  
Sr Protein ◽  
Processing Defects

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.

scholarly journals Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569 ◽  
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

scholarly journals Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing

2010 ◽  
Vol 429 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Marieta Gencheva ◽  
Mitsuo Kato ◽  
Alain N.S. Newo ◽  
Ren-Jang Lin
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Helicase Domain ◽  
Rna Helicases ◽  
Mutant Proteins ◽  
Dead Box ◽  
Nuclear Extracts

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.

scholarly journals Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

2021 ◽  
Author(s):  
Eliezra Glasser ◽  
Debanjana Maji ◽  
Guilia Biancon ◽  
Anees Mohammed Keedakkatt Puthenpeedikakkal ◽  
Chapin Cavender ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Binding ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Central Position ◽  
Nucleotide Substitutions ◽  
Rna Recognition Motifs ◽  
Recognition Motifs ◽  
Polypeptide Backbone ◽  
Dynamics Simulations

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

The stability of dislocation networks under various oxygen-doping conditions in superconducting Bi2Sr2CaCu2Oy single crystals and their influence on the flux pinning properties

1994 ◽  
Vol 52 ◽  
pp. 802-803
Author(s):  
Y. Feng ◽  
X. Y. Cai ◽  
R. J. Kelley ◽  
D. C. Larbalestier
Keyword(s):  
Single Crystals ◽  
Oxygen Content ◽  
Basal Plane ◽  
Flux Pinning ◽  
Pinning Centers ◽  
Before And After ◽  
Anomalous Peak ◽  
Basal Plane Dislocation ◽  
The Stability ◽  
Further Development

The issue of strong flux pinning is crucial to the further development of high critical current density Bi-Sr-Ca-Cu-O (BSCCO) superconductors in conductor-like applications, yet the pinning mechanisms are still much debated. Anomalous peaks in the M-H (magnetization vs. magnetic field) loops are commonly observed in Bi2Sr2CaCu2Oy (Bi-2212) single crystals. Oxygen vacancies may be effective flux pinning centers in BSCCO, as has been found in YBCO. However, it has also been proposed that basal-plane dislocation networks also act as effective pinning centers. Yang et al. proposed that the characteristic scale of the basal-plane dislocation networksmay strongly depend on oxygen content and the anomalous peak in the M-H loop at ˜20-30K may be due tothe flux pinning of decoupled two-dimensional pancake vortices by the dislocation networks. In light of this, we have performed an insitu observation on the dislocation networks precisely at the same region before and after annealing in air, vacuumand oxygen, in order to verify whether the dislocation networks change with varying oxygen content Inall cases, we have not found any noticeable changes in dislocation structure, regardless of the drastic changes in Tc and the anomalous magnetization. Therefore, it does not appear that the anomalous peak in the M-H loops is controlled by the basal-plane dislocation networks.

Sorbitol enhances the physicochemical stability of B12 vitamers

2020 ◽  
Vol 90 (5-6) ◽  
pp. 439-447 ◽  
Author(s):  
Andrew Hadinata Lie ◽  
Maria V Chandra-Hioe ◽  
Jayashree Arcot
Keyword(s):  
Ascorbic Acid ◽  
Uv Light ◽  
Heat Exposure ◽  
Water Soluble ◽  
Uv Exposure ◽  
Physicochemical Stability ◽  
Before And After ◽  
The Stability

Abstract. The stability of B12 vitamers is affected by interaction with other water-soluble vitamins, UV light, heat, and pH. This study compared the degradation losses in cyanocobalamin, hydroxocobalamin and methylcobalamin due to the physicochemical exposure before and after the addition of sorbitol. The degradation losses of cyanocobalamin in the presence of increasing concentrations of thiamin and niacin ranged between 6%-13% and added sorbitol significantly prevented the loss of cyanocobalamin (p<0.05). Hydroxocobalamin and methylcobalamin exhibited degradation losses ranging from 24%–26% and 48%–76%, respectively; added sorbitol significantly minimised the loss to 10% and 20%, respectively (p < 0.05). Methylcobalamin was the most susceptible to degradation when co-existing with ascorbic acid, followed by hydroxocobalamin and cyanocobalamin. The presence of ascorbic acid caused the greatest degradation loss in methylcobalamin (70%-76%), which was minimised to 16% with added sorbitol (p < 0.05). Heat exposure (100 °C, 60 minutes) caused a greater loss of cyanocobalamin (38%) than UV exposure (4%). However, degradation losses in hydroxocobalamin and methylcobalamin due to UV and heat exposures were comparable (>30%). At pH 3, methylcobalamin was the most unstable showing 79% degradation loss, which was down to 12% after sorbitol was added (p < 0.05). The losses of cyanocobalamin at pH 3 and pH 9 (~15%) were prevented by adding sorbitol. Addition of sorbitol to hydroxocobalamin at pH 3 and pH 9 reduced the loss by only 6%. The results showed that cyanocobalamin was the most stable, followed by hydroxocobalamin and methylcobalamin. Added sorbitol was sufficient to significantly enhance the stability of cobalamins against degradative agents and conditions.

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