RNA Duplexes Research Articles

Calculation of Energy for RNA/RNA and DNA/RNA Duplex Formation by Molecular Dynamics Simulation

The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the NA-based constructs. Modern advances in computer modeling methods provide the feasibility of these calculations. We have analyzed the possibility of calculating the energy of DNA/RNA and RNA/RNA duplex formation using representative sets of complexes (65 and 75 complexes, respectively). We used the classical molecular dynamics (MD) method, the MMPBSA or MMGBSA approaches to calculate the enthalpy (ΔH°) component, and the quasi-harmonic approximation (Q-Harm) or the normal mode analysis (NMA) methods to calculate the entropy (ΔS°) contribution to the Gibbs energy (Delta G_{{37}}^{^circ } ) of the NA complex formation. We have found that the MMGBSA method in the analysis of the MD trajectory of only the NA duplex and the empirical linear approximation allow calculation of the enthalpy of formation of the DNA, RNA, and hybrid duplexes of various lengths and GC content with an accuracy of 8.6%. Within each type of complex, the combination of rather efficient MMGBSA and Q-Harm approaches being applied to the trajectory of only the bimolecular complex makes it possible to calculate the Delta G_{{37}}^{^circ } of the duplex formation with an error value of 10%. The high accuracy of predictive calculation for different types of natural complexes (DNA/RNA, DNA/RNA, and RNA/RNA) indicates the possibility of extending the considered approach to analogs and derivatives of nucleic acids, which gives a fundamental opportunity in the future to perform rational design of new types of NA-targeted sequence-specific compounds.

RNA regulation in immunity.

RNA regulation in immunity.

Regulation of GSTu1-mediated insecticide resistance in Plutella xylostella by miRNA and lncRNA.

The evolution of resistance to insecticides is well known to be closely associated with the overexpression of detoxifying enzymes. Although the role of glutathione S-transferase (GST) genes in insecticide resistance has been widely reported, the underlying regulatory mechanisms are poorly understood. Here, one GST gene (GSTu1) and its antisense transcript (lnc-GSTu1-AS) were identified and cloned, and both of them were upregulated in several chlorantraniliprole-resistant Plutella xylostella populations. GSTu1 was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Furthermore, we demonstrated that lnc-GSTu1-AS interacted with GSTu1 by forming an RNA duplex, which masked the binding site of miR-8525-5p at the GSTu1-3′UTR. In summary, we revealed that lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation that could have been induced by miR-8525-5p and thus increased the resistance of P. xylostella to chlorantraniliprole. Our findings reveal a new noncoding RNA-mediated pathway that regulates the expression of detoxifying enzymes in insecticide-resistant insects and offer opportunities for the further understanding of the mechanisms of insecticide and drug resistance.

Contact pairs of RNA with magnesium ions-electrostatics beyond the Poisson-Boltzmann equation

The electrostatic interaction of RNA with its aqueous environment is most relevant for defining macromolecular structure and biological function. The attractive interaction of phosphate groups in the RNA backbone with ions in the water environment leads to the accumulation of positively charged ions in the first few hydration layers around RNA. Electrostatics of this ion atmosphere and the resulting ion concentration profiles have been described by solutions of the nonlinear Poisson-Boltzmann equation and atomistic molecular dynamics (MD) simulations. Much less is known on contact pairs of RNA phosphate groups with ions at the RNA surface, regarding their abundance, molecular geometry, and role in defining RNA structure. Here, we present a combined theoretical and experimental study of interactions of a short RNA duplex with magnesium (Mg2+) ions. MD simulations covering a microsecond time range give detailed hydration geometries as well as electrostatics and spatial arrangements of phosphate-Mg2+ pairs, including both pairs in direct contact and separated by a single water layer. The theoretical predictions are benchmarked by linear infrared absorption and nonlinear two-dimensional infrared spectra of the asymmetric phosphate stretch vibration which probes both local interaction geometries and electric fields. Contact pairs of phosphate groups and Mg2+ ions are identified via their impact on the vibrational frequency position and line shape. A quantitative analysis of infrared spectra for a range of Mg2+-excess concentrations and comparison with fluorescence titration measurements shows that on average 20–30% of the Mg2+ ions interacting with the RNA duplex form contact pairs. The experimental and MD results are in good agreement. In contrast, calculations based on the nonlinear Poisson-Boltzmann equation fail in describing the ion arrangement, molecular electrostatic potential, and local electric field strengths correctly. Our results underline the importance of local electric field mapping and molecular-level simulations to correctly account for the electrostatics at the RNA-water interface.

Overcoming GNA/RNA base-pairing limitations using isonucleotides improves the pharmacodynamic activity of ESC+GalNAc-siRNAs.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

A Dimethylaminophenyl‐Substituted Naphtho[1,2‐b]quinolizinium as a Multicolor NIR Probe for the Fluorimetric Detection of Intracellular Nucleic Acids and Proteins

AbstractThe dye 3‐(4‐(N,N‐dimethylamino)phenyl)naphtho[1,2‐b]quinolizinium was synthesized by means of a Suzuki–Miyaura reaction in good yield, and its binding properties with duplex DNA, quadruplex DNA (G4‐DNA), RNA, and bovine serum albumin (BSA) were investigated by photometric, fluorimetric and polarimetric titrations and DNA denaturation analysis. The compound intercalates into DNA and RNA, associates in binding site I of BSA, and binds to G4‐DNA by terminal π stacking. The ligand exhibits a fluorescence light‐up effect upon complexation to these biomacromolecules, which is more pronounced and blue shifted in the presence of BSA (Φfl=0.29, λfl=627 nm) than with the nucleic acids (Φfl=0.01–0.05, λfl=725–750 nm). Furthermore, the triple‐exponential fluorescence decay of the probe when bound to biomacromolecules in a cell enables their visualization in this medium and the differential labeling of cellular components.

HOXC11 positively regulates the long non-coding RNA HOTAIR and is associated with poor prognosis in colon adenocarcinoma.

Colorectal cancer ranks third in terms of incidence and second in terms of mortality worldwide. The homeobox transcript antisense intergenic RNA (HOTAIR), which was found to be located on the antisense chain of the homeobox C (HOXC) gene cluster, is a long non-coding RNA involved in multiple types of tumors. The role of HOXC11 in tumors remains unclear. Reverse transcription-quantitative PCR was performed to detect the expression level of HOXC11 in colon adenocarcinoma. Cell proliferation and invasion were assessed. RNase protection assay was used to test the possibility of RNA duplex formation. The increased expression and co-expression trend of HOXC11 and HOTAIR were identified in multiple types of cancer from The Cancer Genome Atlas and the results were validated in 12 colon adenocarcinoma and paired non-tumor tissue samples. The expression of HOXC11 and HOTAIR was found to be associated with poor prognosis in colon adenocarcinoma and kidney renal clear cell carcinoma. Furthermore, HOXC11 was found to positively regulate HOTAIR by RNA duplex formation and promoted the proliferation and invasion of colon adenocarcinoma cells.

A new strategy to increase RNA editing at the Q/R site of GluA2 AMPA receptor subunits by targeting alternative splicing patterns of ADAR2

BackgroundThe GluA2 subunit of AMPA receptors (AMPARs) undergoes RNA editing at a specific base mediated by the enzyme ADAR2, changing the coded amino acid from a glutamine to arginine at the so-called Q/R site, which is critical for regulating calcium permeability. ADAR2 exists as multiple alternatively-spliced variants within mammalian cells with differing editing efficiency. New methodIn this study, phosphorodiamidate morpholino oligomers (PMOs) were used to increase Q/R site editing, by affecting the alternative splicing of ADAR2. ResultsPMOs targeting the ADAR2 pre-mRNA transcript successfully induced alternative splicing around the AluJ cassette leading to expression of a more active isoform with increased editing of the GluA2 subunit compared to control. Comparison with existing method(s)Previously PMOs have been used to disrupt RNA editing via steric hindrance of the GluA2 RNA duplex. In contrast we report PMOs that can increase the expression of more catalytically active variants of ADAR2, leading to enhanced GluA2 Q/R RNA editing. ConclusionsUsing PMOs to increase Q/R site editing is presented here as a validated method that would allow investigation of downstream cellular processes implicated in altered ADAR2 activity.

Chimeric Flaviviral RNA-siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells.

RNA is an emerging platform for drug delivery, but the susceptibility of RNA to nuclease degradation remains a major barrier to its implementation in vivo. Here, we engineered flaviviral Xrn1‐resistant RNA (xrRNA) motifs to host small interfering RNA (siRNA) duplexes. The xrRNA‐siRNA molecules self‐assemble in vitro, resist degradation by the conserved eukaryotic 5’ to 3’ exoribonuclease Xrn1, and trigger gene silencing in 293T cells. The resistance of the molecules to Xrn1 does not translate to stability in blood serum. Nevertheless, our results demonstrate that flavivirus‐derived xrRNA motifs can confer Xrn1 resistance on a model therapeutic payload and set the stage for further investigations into using the motifs as building blocks in RNA nanotechnology.

The molecular mechanism of RIG-I activation and signaling.

RIG‐I is our first line of defense against RNA viruses, serving as a pattern recognition receptor that identifies molecular features common among dsRNA and ssRNA viral pathogens. RIG‐I is maintained in an inactive conformation as it samples the cellular space for pathogenic RNAs. Upon encounter with the triphosphorylated terminus of blunt‐ended viral RNA duplexes, the receptor changes conformation and releases a pair of signaling domains (CARDs) that are selectively modified and interact with an adapter protein (MAVS), thereby triggering a signaling cascade that stimulates transcription of interferons. Here, we describe the structural determinants for specific RIG‐I activation by viral RNA, and we describe the strategies by which RIG‐I remains inactivated in the presence of host RNAs. From the initial RNA triggering event to the final stages of interferon expression, we describe the experimental evidence underpinning our working knowledge of RIG‐I signaling. We draw parallels with behavior of related proteins MDA5 and LGP2, describing evolutionary implications of their collective surveillance of the cell. We conclude by describing the cell biology and immunological investigations that will be needed to accurately describe the role of RIG‐I in innate immunity and to provide the necessary foundation for pharmacological manipulation of this important receptor.

Exploring the Nucleobase-Specific Hydrophobic Interaction of Cryptolepine Hydrate with RNA and Its Subsequent Sequestration.

The study of the interactions of drug molecules with genetic materials plays a key role underlying the development of new drugs for many life-threatening diseases in pharmaceutical industries. Understanding their fundamental base-specific and/or groove-binding interaction is crucial to target the genetic material with an external drug, which can pave the way to curing diseases related to the genetic material. Here, we studied the interaction of cryptolepine hydrate (CRYP) with RNA under physiological conditions knowing the antimalarial and anticancer activities of the drug. Our experiments explicitly demonstrate that CRYP interacts with the guanine- and adenine-rich region within the RNA duplex. The pivotal role of the hydrophobic interaction governing the interaction is substantiated by temperature-dependent isothermal titration calorimetry experiments and spectroscopic studies. Circular dichroism study underpins a principally intercalative mode of binding of CRYP with RNA. This interaction is found to be drastically affected in the presence of magnesium salt, which has a strong propensity to coordinate with RNA nucleobases, which can in turn modulate the interaction of the drug with RNA. The temperature-dependent calorimetric results substantiate the occurrence of entropy-enthalpy compensation, which enabled us to rule out the possibility of groove binding of the drug with RNA. Furthermore, our results also show the application of host-guest chemistry in sequestering the RNA-bound drug, which is crucial to the development of safer therapeutic applications.

Developing an Updated Strategy for Estimating the Free-Energy Parameters in RNA Duplexes

For the last 20 years, it has been common lore that the free energy of RNA duplexes formed from canonical Watson–Crick base pairs (bps) can be largely approximated with dinucleotide bp parameters and a few simple corrective constants that are duplex independent. Additionally, the standard benchmark set of duplexes used to generate the parameters were GC-rich in the shorter duplexes and AU-rich in the longer duplexes, and the length of the majority of the duplexes ranged between 6 and 8 bps. We were curious if other models would generate similar results and whether adding longer duplexes of 17 bps would affect the conclusions. We developed a gradient-descent fitting program for obtaining free-energy parameters—the changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS), and the melting temperature (Tm)—directly from the experimental melting curves. Using gradient descent and a genetic algorithm, the duplex melting results were combined with the standard benchmark data to obtain bp parameters. Both the standard (Turner) model and a new model that includes length-dependent terms were tested. Both models could fit the standard benchmark data; however, the new model could handle longer sequences better. We developed an updated strategy for fitting the duplex melting data.

KNOCKDOWN OF Dnmt1 AND Dnmt3a ENZYMES DISRUPTS PREIMPLANTATION EMBRYO DEVELOPMENT BY AFFECTING GLOBAL DNA METHYLATION

DNA methylation is one of the epigenetic mechanisms that play critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of global DNA methylation and Dnmt3a for the de novo methylation, are gradually erased from zygote to blastocyst stage by a demethylation process, and then reestablished by de novo methylation around blastocyst stage. This important differentiation mechanism indicates pivotal roles of global DNA methylation during the course of preimplantation embryo development. So, a fundamental question which also served as the aim of the current study was to address the spatio-temporal expression of Dnmt1 and Dnmt3a and thus the establishment of DNA methylation could affect the embryo development. We set up four groups as control, Dnmt1 siRNA, Dnmt3a siRNA, and non-targeted siRNA. In vivo developed Balb/c mouse zygotes were collected, divided into groups and then cultured for 96 h to reach blastocyst stage. Zygotes were transfected with 50 nM Dnmt1 and Dnmt3a-spesific small interfering RNA (siRNA) duplexes vs non-targeting control siRNA duplexes for 96 h using DharmaFECT as a transfection reagent. Nontargeting siRNA duplex served as the negative control. Knockdown of Dnmt genes using siRNA was confirmed by analyzing the proteins using immunofluorescence and Western blot techniques after 96 h in culture. Following knockdown of Dnmt1 and Dnmt3a, we analyzed the developmental competence of embryos. We also evaluated global DNA methylation levels by 5 methylcytosine staining in blastocyst stage. We found that both Dnmt genes were successfully knocked-down (approximately 80%) in 2-4 cell embryos. In Dnmt1 siRNA group, embryo arrest rates were 29.5% in 2-cell, 15.7 in %4-cell, and 9.8% in morula stages. Only 16.7% embryos reached to blastocyst stage in this group. In Dnmt3a siRNA group, embryos arrested in 26.2% 2-cell, 21.9% 4-cell, 9.2% morula and 29.5% embryos reached to blastocyst stage. Degenerated embryo rates were 28.2% in Dnmt1 siRNA, and 13.1% in Dnmt3a siRNA group. Interestingly, we noted that global DNA methylation level significantly decreased in Dnmt1 knockdown group while increased in Dnmt3a knockdown group (P<0.001). These results suggested that temporal Dnmt enzymes could contribute to the process of early embryonic development and thus play vital roles in the mouse embryos. Therefore it seems evident that these enzymes should also be elucidated in human to check whether they play any major role in the human preimplantation embryo development.

Non-coding RNA regulatory networks in mesothelioma: a narrative review of their implication in innate immune signaling pathways

: Malignant mesothelioma is a rare but rapidly fatal disease highly enriched for innate immunity signature in a subset of patients with better clinical outcome. We provide here an overview of current knowledge on RNA regulatory networks altered in mesothelioma and resulting in increased expression of non-coding-RNA ligands stimulating the innate immune system. The focus on non-coding RNA (ncRNA) ligands is dictated by the fact that a large fraction of the non-coding genome is known to be transcribed and forms duplex RNAs able to stimulate anti-viral defense. Hence, we mostly describe double-stranded RNA sensors such as cytosolic RIG-I-like receptors and endosomal leucine-rich receptors Toll-like receptors 3 and their endogenous ligands. The latter include products downstream alteration of alternative splicing, altered RNA processing or expression of repetitive elements induced by demethylation events or deficiency of chromatin remodelers such as histone methyltransferase SETDB1 or histone demethylase KDM1A/LSD1 or inhibition of CDK4/6. Based on knowledge acquired either in experimental pre-clinical models or in clinical trials in other cancers types, all these events are likely to influence the outcome of mesothelioma patients treatment with new modalities which are explored in current mesothelioma clinical trials. Furthermore, immune checkpoint inhibition became recently standard of care in unresectable mesothelioma.

THE DEAD-BOX PROTEIN CSHA FROM Staphylococcus aureus Mu 50 EXHIBITS RIBONUCLEASE ACTIVITY

DEAD-box proteins play important roles in many RNA processes ranging from RNA synthesis to RNA decay. Furthermore, it has been reported that some bacterial DEAD-box proteins known to be components of the RNA degradosome do not cleave RNA substrates directly. However, the role of DEAD-box proteins in RNA degradation is poorly understood. The present study demonstrated that the DEAD-box protein CshA from the vancomycin-resistant Staphylococcus aureus strain Mu50 possesses RNA degradation activity, ribonuclease activity. Despite having RNA-dependent ATPase activity, CshA did not exhibit RNA helicase activity in vitro. Instead, CshA catalyzed the degradation of single-stranded RNAs of various duplex RNA substrates to form blunt-end RNA products. Thus, we suggest that the ribonuclease activity of the DEAD-box protein CshA may contribute to RNA remodeling in the bacterial RNA degradosome. To our knowledge, this study is the first to report that a DEAD-box protein from a pathogenic bacterium is implicated in multiple ATP-independent activity on RNA, such as degradation.

A Carbazole-Derived Nitroxide That Is an Analogue of Cytidine: A Rigid Spin Label for DNA and RNA.

A variety of semirigid and rigid spin labels comprise a valuable arsenal for measurements of biomolecular structures and dynamics by electron paramagnetic resonance (EPR) spectroscopy. Here, we report the synthesis and characterization of rigid spin labels Ċ and Ċm for DNA and RNA, respectively, that are carbazole-derived nitroxides and analogues of cytidine. Ċ and Ċm were converted to their phosphoramidites and used for their incorporation into oligonucleotides by solid-phase synthesis. Analysis of Ċ and Ċm by single-crystal X-ray crystallography verified their identity and showed little deviation from planarity of the nucleobase. Analysis of the continuous-wave (CW) EPR spectra of the spin-labeled DNA and RNA duplexes confirmed their incorporation into the nucleic acids and the line-shape was characteristic of rigid spin labels. Circular dichroism (CD) and thermal denaturation studies of the Ċ-labeled DNAs and Ċm-labeled RNAs indicated that the labels are nonperturbing of duplex structure.

Insights into the structure and RNA-binding specificity of Caenorhabditis elegans Dicer-related helicase 3 (DRH-3).

DRH-3 is critically involved in germline development and RNA interference (RNAi) facilitated chromosome segregation via the 22G-siRNA pathway in Caenorhabditis elegans. DRH-3 has similar domain architecture to RIG-I-like receptors (RLRs) and belongs to the RIG-I-like RNA helicase family. The molecular understanding of DRH-3 and its function in endogenous RNAi pathways remains elusive. In this study, we solved the crystal structures of the DRH-3 N-terminal domain (NTD) and the C-terminal domains (CTDs) in complex with 5′-triphosphorylated RNAs. The NTD of DRH-3 adopts a distinct fold of tandem caspase activation and recruitment domains (CARDs) structurally similar to the CARDs of RIG-I and MDA5, suggesting a signaling function in the endogenous RNAi biogenesis. The CTD preferentially recognizes 5′-triphosphorylated double-stranded RNAs bearing the typical features of secondary siRNA transcripts. The full-length DRH-3 displays unique structural dynamics upon binding to RNA duplexes that differ from RIG-I or MDA5. These features of DRH-3 showcase the evolutionary divergence of the Dicer and RLR family of helicases.

The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer.

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

Expression of the ISPpu9 transposase of Pseudomonas putida KT2440 is regulated by two small RNAs and the secondary structure of the mRNA 5'-untranslated region.

Insertion sequences (ISs) are mobile genetic elements that only carry the information required for their own transposition. Pseudomonas putida KT2440, a model bacterium, has seven copies of an IS called ISPpu9 inserted into repetitive extragenic palindromic sequences. This work shows that the gene for ISPpu9 transposase, tnp, is regulated by two small RNAs (sRNAs) named Asr9 and Ssr9, which are encoded upstream and downstream of tnp, respectively. The tnp mRNA has a long 5′-untranslated region (5′-UTR) that can fold into a secondary structure that likely includes the ribosome-binding site (RBS). Mutations weakening this structure increased tnp mRNA translation. Asr9, an antisense sRNA complementary to the 5′-UTR, was shown to be very stable. Eliminating Asr9 considerably reduced tnp mRNA translation, suggesting that it helps to unfold this secondary structure, exposing the RBS. Ectopic overproduction of Asr9 increased the transposition frequency of a new ISPpu9 entering the cell by conjugation, suggesting improved tnp expression. Ssr9 has significant complementarity to Asr9 and annealed to it in vitro forming an RNA duplex; this would sequester it and possibly facilitate its degradation. Thus, the antisense Asr9 sRNA likely facilitates tnp expression, improving transposition, while Ssr9 might counteract Asr9, keeping tnp expression low.

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1–4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.