Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex.

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

Identification and validation of N7 -methylguanosine-associated gene NCBP1 as prognostic and immune-associated biomarkers in breast cancer patients.

We intend to evaluate the importance of N7 -methylguanosine (m7G) for the prognosis of breast cancer (BC). We gained 29 m7G-related genes from the published literature and among them, 16 m7G-related genes were found to have differential expression. Five differentially expressed genes (CYFIP1, EIF4E, EIF4E3, NCBP1 and WDR4) were linked to overall survival. This suggests that m7G-related genes might be prognostic or therapeutic targets for BC patients. We put the five genes to LASSO regression analysis to create a four-gene signature, including EIF4E, EIF4E3, WDR4 and NCBP1, that divides samples into two risky groups. Survival was drastically worsened in a high-risk group (p < 0.001). The signature's predictive capacity was demonstrated using ROC (10-year AUC 0.689; 10-year AUC 0.615; 3-year AUC 0.602). We found that immune status was significantly different between the two risk groups. In particular, NCBP1 also has a poor prognosis, with higher diagnostic value in ROC. NCBP1 also has different immune states according to its high or low expression. Meanwhile, knockdown of NCBP1 suppresses BC malignancy in vitro. Therefore, m7G RNA regulators are crucial participants in BC and four-gene mRNA levels are important predictors of prognosis. NCBP1 plays a critical target of m7G mechanism in BC.

The Nuclear Cap-Binding Complex, a multitasking binding partner of RNA polymerase II transcripts.

In eukaryotic cells, RNAs transcribed by RNA polymerase-II receive the modification at the 5' end. This structure is called the cap structure. The cap structure has a fundamental role for translation initiation by recruiting eukaryotic translation initiation factor 4F (eIF4F). The other important mediator of the cap structure is a nuclear cap-binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.

m7G-related genes-NCBP2 and EIF4E3 determine immune contexture in head and neck squamous cell carcinoma by regulating CCL4/CCL5 expression.

Aberrant N7 -methylguanosine (m7G) levels closely correlate with tumor genesis and progression. NCBP2 and EIF4E3 are two important m7G-related cap-binding genes. This study aimed to identify the relationship between the EIF4E3/NCBP2 function and immunological characteristics of head and neck squamous cell carcinoma (HNSCC). Hierarchical clustering was employed in classifying HNSCC patients into two groups based on the expressions of NCBP2 and EIF4E3. The differentially expressed genes were identified between the two groups, and GO functional enrichment was subsequently performed. Weighted gene co-expression network analysis was conducted to identify the hub genes related to EIF4E3/NCBP2 expression and immunity. The differential infiltration of immune cells and the response to immunotherapy were compared between the two groups. Single-cell sequence and trajectory analyses were performed to predict cell differentiation and display the expression of EIF4E3/NCBP2 in each state. In addition, quantitative real-time PCR, spatial transcriptome analysis, transwell assay, and western blotting were conducted to verify the biological function of EIF4E3/NCBP2. Here, group A showed a higher EIF4E3 expression and a lower NCBP2 expression, which had higher immune scores, proportion of most immune cells, immune activities, expression of immunomodulatory targets, and a better response to cancer immunotherapy. Besides, 56 hub molecules with notable immune regulation significance were identified. A risk model containing 17 hub genes and a prognostic nomogram was successfully established. Moreover, HNSCC tissues had a lower EIF4E3 expression and a higher NCBP2 expression than normal tissues. NCBP2 and EIF4E3 played a vital role in the differentiation of monocytes. Furthermore, the expression of CCL4/CCL5 can be regulated via EIF4E3 overexpression and NCBP2 knockdown. Collectively, NCBP2 and EIF4E3 can affect downstream gene expression, as well as immune contexture and response to immunotherapy, which could induce "cold-to-hot" tumor transformation in HNSCC patients.

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins.

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.

The effects of NCBP3 on METTL3-mediated m6A RNA methylation to enhance translation process in hypoxic cardiomyocytes.

Hypoxia as a crucial pathogenesis factor usually results in huge harmful effects on cardiac injury and dysfunction. Our previous study has uncovered the global transcriptome and translatome profiles of cardiomyocytes in vitro and in vivo to response to hypoxia by RNA sequencing and ribosome profiling sequencing. We observe a series of differential expressed genes between transcription and translation, which may be attributed to the hypoxia-specific binding affinity of nuclear cap-binding subunit 3 (NCBP3) at 5' untranslation region of target genes. Although we observe that NCBP3 can facilitate translational process in myocardium under hypoxia stress, the underlying molecular mechanism of NCBP3 for gene translation modulation remains unclear. In this study, we performed NCBP3 immunoprecipitation for mass spectrum and found that METTL3 and eIF4A2 particularly interacted with NCBP3 in hypoxic rat H9C2 cardiomyocytes. Furthermore, we observed that METTL3-mediated N6-methyladenosine (m6A) methylation was elevated in hypoxia, but compromised by NCBP3 or METTL3 knockdown. Finally, we also demonstrated that NCBP3/METTL3/eIF4A2 regulatory axis plays a specific role in cardiomyocytes undergoing hypoxic stress. Taken together, we unmasked NCBP3, a novel hypoxia-specific response protein functions as a scaffold to coordinate METTL3 and eIF4A2 for enhancing gene translation by m6A RNA methylation in cardiomyocytes upon hypoxic stress.

Translation mediated by the nuclear cap-binding complex is confined to the perinuclear region via a CTIF-DDX19B interaction.

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.

NCBP3 positively impacts mRNA biogenesis.

The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

Affinity proteomic dissection of the human nuclear cap-binding complex interactome.

A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed due to its participation in RNA-fate decisions via interactions with RNA productive factors as well as with adapters of the degradative RNA exosome. A novel cap-binding protein, NCBP3, was recently proposed to form an alternative CBC together with NCBP1, and to interact with the canonical CBC along with the protein SRRT. The theme of post-transcriptional RNA fate, and how it relates to co-transcriptional ribonucleoprotein assembly, is abundant with complicated, ambiguous, and likely incomplete models. In an effort to clarify the compositions of NCBP1-, 2- and 3-related macromolecular assemblies, we have applied an affinity capture-based interactome screen where the experimental design and data processing have been modified to quantitatively identify interactome differences between targets under a range of experimental conditions. This study generated a comprehensive view of NCBP-protein interactions in the ribonucleoprotein context and demonstrates the potential of our approach to benefit the interpretation of complex biological pathways.

Mapping domains of ARS2 critical for its RNA decay capacity.

ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.

NCBP2 modulates neurodevelopmental defects of the 3q29 deletion in Drosophila and Xenopus laevis models.

The 1.6 Mbp deletion on chromosome 3q29 is associated with a range of neurodevelopmental disorders, including schizophrenia, autism, microcephaly, and intellectual disability. Despite its importance towards neurodevelopment, the role of individual genes, genetic interactions, and disrupted biological mechanisms underlying the deletion have not been thoroughly characterized. Here, we used quantitative methods to assay Drosophila melanogaster and Xenopus laevis models with tissue-specific individual and pairwise knockdown of 14 homologs of genes within the 3q29 region. We identified developmental, cellular, and neuronal phenotypes for multiple homologs of 3q29 genes, potentially due to altered apoptosis and cell cycle mechanisms during development. Using the fly eye, we screened for 314 pairwise knockdowns of homologs of 3q29 genes and identified 44 interactions between pairs of homologs and 34 interactions with other neurodevelopmental genes. Interestingly, NCBP2 homologs in Drosophila (Cbp20) and X. laevis (ncbp2) enhanced the phenotypes of homologs of the other 3q29 genes, leading to significant increases in apoptosis that disrupted cellular organization and brain morphology. These cellular and neuronal defects were rescued with overexpression of the apoptosis inhibitors Diap1 and xiap in both models, suggesting that apoptosis is one of several potential biological mechanisms disrupted by the deletion. NCBP2 was also highly connected to other 3q29 genes in a human brain-specific interaction network, providing support for the relevance of our results towards the human deletion. Overall, our study suggests that NCBP2-mediated genetic interactions within the 3q29 region disrupt apoptosis and cell cycle mechanisms during development.

NCBP1 promotes the development of lung adenocarcinoma through up-regulation of CUL4B.

Lung cancer is the most frequent cancer type and is the leading cause of tumour-associated deaths worldwide. Nuclear cap-binding protein 1 (NCBP1) is necessary for capped RNA processing and intracellular localization. It has been reported that silencing of NCBP1 resulted in cell growth reduction in HeLa cells. Nevertheless, its clinical significance and underlying molecular mechanisms in non-small-cell lung cancer remain unclear. In this study, we found that NCBP1 was significantly overexpressed in lung cancer tissues and several lung cancer cell lines. Through knockdown and overexpression experiments, we showed that NCBP1 promoted lung cancer cell growth, wound healing ability, migration and epithelial-mesenchymal transition. Mechanistically, we found that cullin 4B (CUL4B) was a downstream target gene of NCBP1 in NSCLC. NCBP1 up-regulated CUL4B expression via interaction with nuclear cap-binding protein 3 (NCBP3). CUL4B silencing significantly reversed NCBP1-induced tumorigenesis in vitro. Based on these findings, we propose a model involving the NCBP1-NCBP3-CUL4B oncoprotein axis, providing novel insight into how CUL4B is activated and contributes to LUAD progression.

Staufen1 and UPF1 exert opposite actions on the replacement of the nuclear cap-binding complex by eIF4E at the 5' end of mRNAs.

Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.

Paip2 cooperates with Cbp80 at an active promoter and participates in RNA Polymerase II phosphorylation in Drosophila.

The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300-kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap-binding complex, with both proteins ensuring proper Pol II C-terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation.

Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export.

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive-sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA.

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.

Properties of the ternary complex formed by yeast eIF4E, p20 and mRNA.

Yeast p20 is a small, acidic protein that binds eIF4E, the cap-binding protein. It has been proposed to affect mRNA translation and degradation, however p20's function as an eIF4E-binding protein (4E-BP) and its physiological significance has not been clearly established. In this paper we present data demonstrating that p20 is capable of binding directly to mRNA due to electrostatic interaction of a stretch of arginine and histidine residues in the protein with negatively charged phosphates in the mRNA backbone. This interaction contributes to formation of a ternary eIF4E/p20/capped mRNA complex that is more stable than complexes composed of capped mRNA bound to eIF4E in the absence of p20. eIF4E/p20 complex was found to have a more pronounced stimulatory effect on capped mRNA translation than purified eIF4E alone. Addition of peptides containing the eIF4E-binding domains present in p20 (motif  YTIDELF), in eIF4G (motif  YGPTFLL) or Eap1 (motif  YSMNELY) completely inhibited eIF4E-dependent capped mRNA translation (in vitro), but had a greatly reduced inhibitory effect when eIF4E/p20 complex was present. We propose that the eIF4E/p20/mRNA complex serves as a stable depository of mRNAs existing in a dynamic equilibrium with other complexes such as eIF4E/eIF4G (required for translation) and eIF4E/Eap1 (required for mRNA degradation).

Structural analysis of human ARS2 as a platform for co-transcriptional RNA sorting.

ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC), it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here, we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC-ARS2-NCBP3 form a ternary complex that is mutually exclusive with CBC-ARS-PHAX (involved in snRNA export). Our results support that mutually exclusive higher-order CBC-ARS2 complexes are critical in determining Pol II transcript fate.

Transcriptional coactivator PGC-1α contains a novel CBP80-binding motif that orchestrates efficient target gene expression.

Although peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a well-established transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, the molecular mechanism by which it functions is incompletely understood. Here we used in vitro binding assays, X-ray crystallography, and immunoprecipitations of mouse myoblast cell lysates to define a previously unknown cap-binding protein 80 (CBP80)-binding motif (CBM) in the C terminus of PGC-1α. We show that the CBM, which consists of a nine-amino-acid α helix, is critical for the association of PGC-1α with CBP80 at the 5' cap of target transcripts. Results from RNA sequencing demonstrate that the PGC-1α CBM promotes RNA synthesis from promyogenic genes. Our findings reveal a new conduit between DNA-associated and RNA-associated proteins that functions in a cap-binding protein surveillance mechanism, without which efficient differentiation of myoblasts to myotubes fails to occur.