Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum.

Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.

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.

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.

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.

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.

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).

Characterizing ZC3H18, a Multi-domain Protein at the Interface of RNA Production and Destruction Decisions.

Nuclear RNA metabolism is influenced by protein complexes connecting to both RNA-productive and -destructive pathways. The ZC3H18 protein binds the cap-binding complex (CBC), universally present on capped RNAs, while also associating with the nuclear exosome targeting (NEXT) complex, linking to RNA decay. To dissect ZC3H18 function, we conducted interaction screening and mutagenesis of the protein, which revealed a phosphorylation-dependent isoform. Surprisingly, the modified region of ZC3H18 associates with core histone proteins. Further examination of ZC3H18 function, by genome-wide analyses, demonstrated its impact on transcription of a subset of protein-coding genes. This activity requires the CBC-interacting domain of the protein, with some genes being also dependent on the NEXT- and/or histone-interacting domains. Our data shed light on the domain requirements of a protein positioned centrally in nuclear RNA metabolism, and they suggest that post-translational modification may modulate its function.

The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5' end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.

Cbp80 is needed for the expression of piRNA components and piRNAs.

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.

Translation initiation mediated by nuclear cap-binding protein complex.

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

Genome-Wide Mapping of Uncapped and Cleaved Transcripts Reveals a Role for the Nuclear mRNA Cap-Binding Complex in Cotranslational RNA Decay in Arabidopsis.

RNA turnover is necessary for controlling proper mRNA levels posttranscriptionally. In general, RNA degradation is via exoribonucleases that degrade RNA either from the 5' end to the 3' end, such as XRN4, or in the opposite direction by the multisubunit exosome complex. Here, we use genome-wide mapping of uncapped and cleaved transcripts to reveal the global landscape of cotranslational mRNA decay in the Arabidopsis thaliana transcriptome. We found that this process leaves a clear three nucleotide periodicity in open reading frames. This pattern of cotranslational degradation is especially evident near the ends of open reading frames, where we observe accumulation of cleavage events focused 16 to 17 nucleotides upstream of the stop codon because of ribosomal pausing during translation termination. Following treatment of Arabidopsis plants with the translation inhibitor cycloheximide, cleavage events accumulate 13 to 14 nucleotides upstream of the start codon where initiating ribosomes have been stalled with these sequences in their P site. Further analysis in xrn4 mutant plants indicates that cotranslational RNA decay is XRN4 dependent. Additionally, studies in plants lacking CAP BINDING PROTEIN80/ABA HYPERSENSITIVE1, the largest subunit of the nuclear mRNA cap binding complex, reveal a role for this protein in cotranslational decay. In total, our results demonstrate the global prevalence and features of cotranslational RNA decay in a plant transcriptome.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.

[Interactions of the TREX-2 complex with mRNP particle of ß-tubulin 56D gene].

mRNA transport from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. A pre-mRNA molecule undergoes modification, such as 5'-capping, splicing, and 3'-end processing, in the nucleus. The molecule being modified interacts with a large number of proteins and, thus, mRNP particles are formed. The binding of factors involved in nuclear export also occurs during transcription and mRNA processing. We have shown that the functioning of TREX-2, an mRNA export complex, is restricted to the nucleus. We used the method of RNA coprecipitation that enables the selective extraction of RNA-protein complexes from samples to show that the transcription elongation complex TREX interacts with mRNA of the ß-tubulin 56D gene over the entire length of the molecule. The capping protein Cbp80 reacted both with the cap structure and with a specific part of the coding mRNA of the ß-tubulin 56D gene. The TREX-2 complex that mediates mRNA export from the nucleus to the cytoplasm is bound to the same part of the coding sequence. Thus, we identified a common binding site for all of the complexes under investigation on the mRNA of ß-tubulin 56D. Co-immunoprecipitation reactions performed with S2 cell extracts revealed interactions between the components of complexes involved in transcription elongation, maturation, and export of mRNA. The model of molecular folding for the mRNP particle involving the mRNA of ß-tubulin 56D has been proposed.