Formation Of RNA-protein Complexes Research Articles

Water-mediated ribonucleotide-amino acid pairs and higher-order structures at the RNA-protein interface: analysis of the crystal structure database and a topological classification.

Water is essential for the formation, stability and function of RNA-protein complexes. To delineate the structural role of water molecules in shaping the interactions between RNA and proteins, we comprehensively analyzed a dataset of 329 crystal structures of these complexes to identify water-mediated hydrogen-bonded contacts at RNA-protein interface. Our survey identified a total of 4963 water bridges. We then employed a graph theory-based approach to present a robust classification scheme, encompassing triplets, quartets and quintet bridging topologies, each further delineated into sub-topologies. The frequency of water bridges within each topology decreases with the increasing degree of water node, with simple triplet water bridges outnumbering the higher-order topologies. Overall, this analysis demonstrates the variety of water-mediated interactions and highlights the importance of water as not only the medium but also the organizing principle underlying biomolecular interactions. Further, our study emphasizes the functional significance of water-mediated interactions in RNA-protein complexes, and paving the way for exploring how these interactions operate in complex biological environments. Altogether, this understanding not only enhances insights into biomolecular dynamics but also informs the rational design of RNA-protein complexes, providing a framework for potential applications in biotechnology and therapeutics. All the scripts, and data are available at https://github.com/PSCPU/waterbridges.

Assembly of the bacterial ribosome with circularly permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the structural insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of 23 resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Internal Normal Mode Analysis Applied to RNA Flexibility and Conformational Changes.

We investigated the capability of internal normal modes to reproduce RNA flexibility and predict observed RNA conformational changes and, notably, those induced by the formation of RNA-protein and RNA-ligand complexes. Here, we extended our iNMA approach developed for proteins to study RNA molecules using a simplified representation of the RNA structure and its potential energy. Three data sets were also created to investigate different aspects. Despite all the approximations, our study shows that iNMA is a suitable method to take into account RNA flexibility and describe its conformational changes opening the route to its applicability in any integrative approach where these properties are crucial.

Abstract 1533: Long non-coding RNA control of cancer cell growth

Abstract Long non-coding RNAs (lncRNAs) are involved in controlling regulatory networks critical for gene expression, cellular growth, and development. Altered expression of lncRNAs are associated with tumor progression in multiple types of human cancers, but the mechanisms by which lncRNAs may control growth and proliferation in cancer cells remain unknown in most cases. Development of interventions targeting non-coding RNA regulation of cell growth would open new avenues for cancer treatment. To identify growth regulatory pathways controlled by non-coding RNAs, we used proteomics and genomics tools to study the effect of changes in lncRNA expression levels in human cancer cell lines. First, we identified five growth regulator lncRNAs based on data mining of CRISPR/Cas9 screens and high throughput sequencing studies from patient tumor and normal cells. These lncRNAs have been associated with progression and metastasis in breast, lung and colon cancers. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. Next, we evaluated the cellular growth phenotype for each lncRNA in overexpression and knockdown strains using multiple established cancer cell lines. We analyzed cellular transcriptome changes after lncRNA perturbation using high-throughput RNA sequencing. Finally, RNA-protein interactions for each growth regulator lncRNA were identified using an RNA hybridization capture method paired with mass spectrometry that enables purification of direct and specific endogenous RNA-binding proteins. Using this combination of transcriptomics and proteomics data, we discovered that perturbation of growth regulator lncRNAs results in dysregulation of growth signaling pathways and increased expression of p53. We are currently identifying the sequence and structure determinants of RNA-protein complex formation to uncover the mechanisms of action of lncRNAs in controlling cancer cell growth. Citation Format: Tong Su, Bobby Kong, Calvin Huang, Jonathan Zhu, Colleen McHugh. Long non-coding RNA control of cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1533.

Long non-coding RNA TINCR promotes hepatocellular carcinoma proliferation and invasion via STAT3 signaling by direct interacting with T-cell protein tyrosine phosphatase (TCPTP)

ABSTRACT The long non-coding RNAs (lncRNAs) participate in modulating numerous important cancer phenotypes via formation of RNA-protein complex. TINCR (terminal differentiation-induced lncRNA) modulates cancer cell behavior in many human malignancies, such as hepatocellular carcinoma (HCC). Herein, we proposed to investigate the underlying mechanism by which TINCR regulates HCC progression via formation of RNA-protein. RNA pulldown, LC-MS/MS, bioinformatics analysis, and RNA immunoprecipitation (RIP) assays were employed to identify TINCR-interacting protein TCPTP in HCC cells. The siRNAs for TINCR and TCPTP were transfected into HCC cells. The plasmids encoding full length or the 1–360 nt deletion of TINCR were generated and applied to cell transfection. The CCK-8, colony formation, EdU, wound healing along with transwell assays were employed to examine cell proliferation, apoptosis, migration, and infiltration. Real-time PCR, as well as western blot assays were employed to assess the levels of STAT3 phosphorylation and its target genes. We identified 1–360 nt region of TINCR, which directly bound with the phosphatase domain of TCPTP to inhibit its tyrosine phosphatase activity. Then, the results showed that the increasing of cell growth, migration, infiltration, and the reducing of apoptosis in TINCR-knockdown HCC cells was remarkably reversed with TCPTP silence. Additionally, Δ1-360 TINCR overexpression did not affect HCC cell growth, apoptosis, migration, infiltration, and STAT3 target genes expression. Our data revealed that TINCR directly bound TCPTP and suppressed the dephosphorylation of STAT3, thus promoting STAT3 activation and its downstream target genes in HCC progression and tumorigenicity. Highlights LncRNA TINCR interacted with protein TCPTP LncRNA TINCR maintained STAT3 phosphorylation LncRNA TINCR affected STAT3 signaling in HCC Abbreviations: lncRNAs: long non-coding RNAs; TINCR: terminal differentiation-induced lncRNA; TCPTP: T cell protein tyrosine phosphatase; siRNA: small-interfering RNA; HCC: hepatocellular carcinoma; nt: nucleotide; LC-MS/MS: Liquid Chromatography - Tandem Mass Spectrometry; RIP: RNA immunoprecipitation; ANOVA: analysis of variance; EdU: 5-ethynyl-2’-deoxyuridine; real-time PCR: real-time polymerase chain reaction; CCK-8: cell counting kit-8; aa: amino acids; STAT3: signal transducer and activator of transcription 3

Demonstration that Small Molecules can Bind and Stabilize Low-abundance Short-lived RNA Excited Conformational States

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.

Fast and unbiased purification of RNA-protein complexes after UV cross-linking.

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 h) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

Detection of RNA-protein interactions using a highly sensitive non-radioactive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) is a sensitive technique useful in the identification and characterization of protein interactors with nucleic acids. This assay provides an efficient method to study DNA or RNA binding proteins and to identify nucleic acid substrates. The specific interaction plays important roles in many biological processes such as transcription, translation, splicing, and global gene expression. In this article, we have modified the EMSA technique and developed a non-radioactive straightforward method to study and determine RNA-protein interactions. The labeling of target RNAs by 3'-end biotinylation and the detection of biotin reactivity to streptavidin-conjugated horseradish peroxidase is a highly sensitive approach capable to detect the formation of RNA-protein complexes. Overall, we provide a complete technical guide useful to determine in vitro RNA-protein interactions and analyze RNA target specificity.

M⁶A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins.

The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m6A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins.

SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation.

Synchronisation of translation rates between the mitochondria and their cellular host is critical for the maintenance of cellular fitness, with cancer cells being especially vulnerable to translation uncoupling. Although alterations of cytosolic protein synthesis are common in human cancer, the compensating mechanisms in place in the mitochondria remain elusive. Here we show that the malignant lncRNA SAMMSON promotes a balanced increase in rRNA maturation and protein synthesis in the cytosol and mitochondria by modulating the localisation of CARF, an RNA-binding protein sequestering XRN2 in the nucleoplasm and limiting nucleolar rRNA maturation. SAMMSON interferes with XRN2 binding to CARF in the nucleus by favouring the formation of an aberrant cytoplasmic RNA-protein complex containing CARF and p32, a mitochondrial protein required for the processing of the mitochondrial rRNAs. This data highlights how a single oncogenic lncRNA can simultaneously modulate RNA-protein complex formation in two distinct cellular compartments to promote cell growth.

Haplotype-specific MAPT exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

BackgroundGenome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium.MethodsIn this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay.ResultsUsing whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion.ConclusionsWe identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium.

Modeling of Protein-RNA Complex Structures Using Computational Docking Methods.

A significant part of biology involves the formation of RNA-protein complexes. X-ray crystallography has added a few solved RNA-protein complexes to the repertoire; however, it remains challenging to capture these complexes and often only the unbound structures are available. This has inspired a growing interest in finding ways to predict these RNA-protein complexes. In this study, we show ways to approach this problem by computational docking methods, either with a fully automated NPDock server or with a workflow of methods for generation of many alternative structures followed by selection of the most likely solution. We show that by introducing experimental information, the structure of the bound complex is rendered far more likely to be within reach. This study is meant to help the user of docking software understand how to grapple with a typical realistic problem in RNA-protein docking, understand what to expect in the way of difficulties, and recognize the current limitations.

A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

Ribosome RNA assembly intermediates visualized in living cells.

In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes.

C. elegans sirtuin SIR-2.4 and its mammalian homolog SIRT6 in stress response.

Stress is a significant life event. The immediate response to stress is critical for survival. In organisms ranging from the unicellular Saccharomyces cerevisiae to protozoa (Trypanosoma brucei) and metazoan (such as Caenorhabditis elegans, Homo sapiens) stress response leads to the formation of cytoplasmic RNA-protein complexes referred to as stress granules (SGs). SGs regulate cell survival during stress by the sequestration of the signaling molecules implicated in apoptosis. They are a transient place of messenger ribonucleoproteins (mRNPs) remodeling for storage, degradation, or reinitiation of translation during stress and recovery from stress. Recently, we have identified chromatin factor, the sirtuin C. elegans SIR-2.4 variant and its mammalian homolog SIRT6 as a regulator of SGs formation. SIRT6 is highly conserved NAD(+)-dependent lysine deacetylase and ADP-ribosyltransferase impacting longevity, metabolism, and cancer. We observed that the cellular formation of SGs by SIRT6 or SIR-2.4 was linked with the cell viability or C. elegans survival and was dependent on SIRT6 enzymatic activity. Here, we discuss how SIR-2.4/SIRT6 influences SGs formation and stress response. We suggest possible mechanisms for such an unanticipated function of a chromatin regulatory factor SIRT6 in assembly of stress granules and cellular stress resistance.

Modulation of RNase E Activity by Alternative RNA Binding Sites

Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5′-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.

Structural Basis of Assembly Chaperone- Mediated snRNP Formation

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

Poly(A) Binding Protein C1 Is Essential for Efficient L1 Retrotransposition and Affects L1 RNP Formation

Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell.

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5’ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme’s secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. However, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart: two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes. Only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.

A cis-acting region in the N-methyl-d-aspartate R1 3′-untranslated region interacts with the novel RNA-binding proteins beta subunit of alpha glucosidase II and annexin A2 - effect of chronic ethanol exposure in vivo

A cis-acting region, Δ4, located in the 3'-untranslated region of N-methyl-d-aspartate R1(NR1) mRNA interacts with several trans-acting proteins present in polysomes purified from fetal cortical neurons. Chronic ethanol exposure of fetal cortical neurons increases Δ4 RNA-protein interactions. This increased interaction is due to an increase in one of the Δ4-binding trans-acting proteins identified as beta subunit of alpha glucosidase II (GIIβ). In this study, we examined whether ethanol-mediated regulation of NR1 mRNA in vivo is similar to that in vitro and whether Δ4-trans interactions are important for ethanol-mediated NR1 mRNA stability. Our data show that polysomal proteins from adult mouse cerebral cortex (CC) formed a complex with Δ4 RNA, suggesting the presence of NR1 mRNA-binding trans-acting proteins in CC polysomes. The intensity of the Δ4 RNA-protein complex was increased with polysomes from chronic ethanol-exposed CC. The Δ4 RNA-protein complex harbored GIIβ and a second trans-acting protein identified as annexin A2 (AnxA2). Ethanol-sensitive GIIβ was upregulated by 70% in ethanol-exposed CC. Heparin, a known binding partner of AnxA2, inhibited Δ4 RNA-protein complex formation. Transient transfection studies using chimeric constructs with and without the Δ4 region revealed that cis-trans interactions are important for ethanol-mediated stability of NR1 mRNA. Furthermore, our data highlight, for the first time, the presence of a binding site on the 3'-untranslated region of NR1 mRNA for AnxA2 and demonstrate the regulation of NR1 mRNA by AnxA2, GIIβ and a third NR1 mRNA-binding protein, which is yet to be identified.