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. 2024 Mar 20;47(4):100049. doi: 10.1016/j.mocell.2024.100049

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

Min-Kyung Shin 1, Jeeyoon Chang 2, Joori Park 1, Hyuk-Joon Lee 1, Jae-Sung Woo 1,⁎, Yoon Ki Kim 2,⁎
PMCID: PMC11016901  PMID: 38513766

Abstract

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.

Keywords: Cap-binding complex, Endoplasmic reticulum, Eukaryotic translation initiation factor 4E, Nonsense-mediated messenger RNA decay, Signal recognition particle

INTRODUCTION

In the nucleus of eukaryotic cells, the nuclear cap-binding complex (CBC), composed of cap-binding protein (CBP) 80 and CBP 20, recognizes the 5′-cap structure of newly synthesized precursor forms of mRNAs (Maquat et al., 2010, Ryu and Kim, 2017). After additional processing, including splicing and 3′-polyadenylation, the messenger ribonucleoproteins (mRNPs) are exported from the nucleus to the cytoplasm through the nuclear pore complex with their 5′-cap bound by CBC (Maquat et al., 2010, Ryu and Kim, 2017). During or after mRNP export, the CBC recruits the ribosome and drives the first (or pioneer) round of translation (Chiu et al., 2004, Ishigaki et al., 2001, Maquat et al., 2010, Ryu and Kim, 2017). During the pioneer round of translation, the mRNP undergoes dramatic remodeling. Then, the CBC at the 5′-cap structure of the mRNA is replaced by the cytoplasmic eukaryotic translation initiation factor 4E (eIF4E). eIF4E-associated mRNA is used as a template for multiple rounds of translation, known as steady-state translation (Chiu et al., 2004, Maquat et al., 2010, Ryu and Kim, 2017). Although the CBC and eIF4E share a common ability to recruit ribosomes in the cytoplasm and drive pioneer and steady-state translation, respectively, their molecular functions and cellular roles differ. CBC-driven pioneer translation is coupled mainly with the mRNA surveillance pathway, typified by nonsense-mediated mRNA decay (NMD) (Kim and Krainer, 2023, Kim and Maquat, 2019, Kurosaki et al., 2019, Kwon et al., 2023, Popp and Maquat, 2014). In contrast, eIF4E-driven steady-state translation is involved in the synthesis of a large quantity of proteins.

NMD is the best-characterized mRNA quality control pathway in eukaryotic cells, by which aberrant or faulty mRNAs harboring premature termination codons (PTCs) are selectively recognized and their entire sequences are rapidly degraded before the expression of potentially deleterious and toxic truncated polypeptides (Kim and Maquat, 2019, Kurosaki et al., 2019, Kwon et al., 2023, Popp and Maquat, 2014). During the pioneer translation of PTC-containing mRNA, the ribosome reaching the PTC associates with up-frameshift protein 1 (UPF1). In this circumstance, the presence of NMD-stimulating features, such as an exon junction complex deposited onto each exon-exon junction after a splicing event in the nucleus (Jung and Kim, 2023, Le Hir et al., 2016, Schlautmann and Gehring, 2020), can activate and phosphorylate UPF1 by suppressor of morphogenesis in genitalia 1 (SMG1) kinase. Activated UPF1 recruits several decay-inducing enzymes or adapters, including SMG5-7 and proline-rich nuclear receptor coactivator 2, consequently leading to rapid degradation of the whole body of PTC-containing mRNA (Kim and Maquat, 2019, Kurosaki et al., 2019). It should be noted that whereas NMD is primarily coupled to CBC-driven pioneer translation, eIF4E-bound mRNAs can also be targeted for NMD (Durand and Lykke-Andersen, 2013, Popp and Maquat, 2014, Rufener and Muhlemann, 2013). The molecular features that dictate NMD on either CBC-associated or eIF4E-associated mRNAs remain unknown.

A recent study unravels an additional crucial role of the pioneer translation in the proper targeting of mRNAs encoding a signal peptide to the endoplasmic reticulum (ER) (Park et al., 2021). In the case that mRNA encodes integral membrane protein or secreted protein, the nascent signal peptide synthesized by the pioneer translation from the CBC-bound mRNA is transiently captured by a signal recognition particle (SRP) composed of 7SL noncoding RNA and 6 polypeptides (Aviram and Schuldiner, 2017, Chartron et al., 2016, Kobayashi et al., 2018, Mercier et al., 2017, Rapoport et al., 2017, Schibich et al., 2016). This interaction causes the complex composed of the CBC, ribosome-nascent chain complex (RNC), and SRP to be translationally arrested. Concomitantly, SRP54, a component of SRP, directly associates with CBP80, blocking the accessibility of importin-β (IMPβ), which promotes the replacement of CBC by eIF4E (Park et al., 2021). As a result, the CBC-RNC-SRP complex remains a translationally inactive complex with the 5′-cap of mRNA bound by CBC.

When the CBC-RNC-SRP complex is properly delivered to the surface of the ER, it interacts with the SRP receptor (SR), a heterodimer of SRα and SRβ located on the ER surface. The interaction between SR and SRP disrupts the interaction between CBP80 and SRP54, allowing for the conversion from CBC-RNC-SRP to eIF4E-RNC-SRP by IMPβ and releasing the translational arrest (Park et al., 2021). Consequently, the remaining region of the mRNA with its 5′-cap bound by eIF4E can be translated (Aviram and Schuldiner, 2017, Chartron et al., 2016, Kobayashi et al., 2018, Mercier et al., 2017, Rapoport et al., 2017, Schibich et al., 2016). In this way, the CBC-driven pioneer translation is involved in the translational repression of RNC-SRP before the ER delivery of the RNC-SRP. This phenomenon is called CBC-ensured translational repression of RNC-SRP (CENTRE) (Park et al., 2021). When the CENTRE is inefficient or the interaction of CBP80 and SRP54 is disrupted before the ER delivery of the RNC-SRP, IMPβ would easily access CBC-RNC-SRP and promote its conversion into eIF4E-RNC-SRP, which may lead to polypeptide synthesis occurring at inappropriate cellular locations. These events cause the accumulation of aberrant or misfolded polypeptides in inappropriate cellular locations, triggering a cytosolic stress response associated with many neurodegenerative diseases (Hipp et al., 2019, Sontag et al., 2017).

In this study, we demonstrate that CENTRE occurs at the transcriptome-wide level to ensure the proper expression of mRNAs encoding signal peptides. Moreover, the NMD of mRNAs encoding a signal peptide is repressed by CENTRE until the mRNAs are targeted to the ER surface, indicating the functional importance of CENTRE in the process of both proper targeting of the CBC-RNC-SRP complex to the ER and mRNA surveillance.

MATERIALS AND METHODS

Cell Culture

HeLa (female; American type culture collection) and HEK293T (fetal; American type culture collection) cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin solution (Sigma-Aldrich) at 37 °C and 5% CO2. The cells were frequently treated with plasmocin (InvivoGen) to minimize mycoplasma contamination. Mycoplasma contamination was assessed using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza).

Plasmid Construction

The following plasmids were used in this study: phCMV-MUP (Kim et al., 2005); pCon-RβGl-Norm and pPPL-RβGl-Norm (Park et al., 2021); pRβGl-SL0-Ter (Choe et al., 2014); and pWT-PPL-TET (Karamyshev et al., 2014).

To generate plasmids pPPL-RβGl-Ter and pCon-RβGl-Ter, preprolactin (PPL) and its control (Con) sequences from pPPL-RβGl-Norm or pCon-RβGl-Norm were inserted into pRβGl-SL0-Ter, respectively.

All polymerase chain reactions (PCRs) were conducted using an Advantage-HF2 PCR Kit (Clontech). All constructs were confirmed using sequencing.

DNA or small interfering RNA Transfection

Cells were transiently transfected with various plasmids using Lipofectamine 2000 (Invitrogen) or 100 nM in vitro-synthesized small interfering RNAs (siRNAs) (Bioneer) using Lipofectamine 3000 (Invitrogen). For immunoprecipitation (IP) experiments, the indicated plasmids were transiently transfected into HEK293T cells using the calcium phosphate transfection method.

The specific downregulation of endogenous proteins was accomplished using the following siRNA sequences: 5′-r(AAAUUUGGUAAGAACAAUG)d(TT)-3′ (SRα siRNA), 5′-r(UAUUGUUGACUCUGUAUAC)d(TT)-3′ (SRβ siRNA), 5′-r(GGAAGAAGCUAAAGAGAAA)d(TT)-3′ (CBP80 siRNA), r(GAUGCAGUUCCGCUCCAUU)d(TT)-3′ (UPF1 siRNA), and 5′-r(ACAAUCCUGAUCAGAAACC)d(TT)-3′ (nonspecific control siRNA).

RNA Preparation, Reverse Transcription Polymerase Chain Reaction (RT-PCR), and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Total-cells RNAs purified with TRIzol reagent (Life Technologies) were subjected to DNA digestion using 0.05 U/μL DNase I (Thermo Fischer Scientific) supplemented with 0.4 U/μL RNase inhibitor (Thermo Scientific) at 37 °C for 45 min. The purified RNA was incubated with 6 U/μL RevertAid reverse transcriptase (Thermo Fisher Scientific) supplemented with 1 U/μL RNase inhibitor at 37 °C for 2 h. Then, qRT-PCR analyses were performed with TOPreal qPCR 2× PreMIX (Enzynomics) and gene-specific oligonucleotides on a LightCycler 480 SYBR Green I Master Kit (Roche) according to the messenger ribonucleic acid (MIQE) guidelines (Bustin et al., 2009). The gene-specific oligonucleotide sequences used in this study are listed in Supplementary Table S1.

Measurement of the mRNA Half-life

One day after the transfection of HeLa cells with a specific siRNA, the cells were transfected with the indicated plasmids. Two days later, the cells were treated with 100 μg/mL 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (a potent transcription inhibitor; Sigma-Aldrich) and harvested at various time points. As described above, total RNA was purified and analyzed using qRT-PCR.

Antibodies

Primary antibodies against the following proteins were used for western blotting, IP, or RNA-IP: CBP80 and UPF1 (Cho et al., 2012), eIF4E ([Jeong et al., 2019] for IP; 610269, BD Biosciences for western blotting), SRα (H00006734-B02P, Novus Biologicals), SRβ (NBP2-02028, Novus Biologicals), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; LF-PA0212, Ab Frontier), and β-actin (A5441, Sigma-Aldrich).

The following secondary antibodies conjugated with horseradish peroxidase (HRP) were used for western blotting: a HRP-conjugated goat α-mouse IgG antibody (AP124P, Sigma-Aldrich) and an HRP-conjugated goat α-rabbit IgG antibody (AP132P, Sigma-Aldrich).

IP and RNA-IP

The cells were harvested after washing with ice-cold phosphate-buffered saline. Then, the cells were resuspended using nuclear extraction buffer-2 (NET-2) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Sigma-Aldrich], 2 mM benzamidine hydrochloride [Sigma-Aldrich], 0.05% NP-40 [IGEPAL CA-630; Sigma-Aldrich], 10 mM sodium fluoride [Sigma-Aldrich], and 0.25 mM sodium orthovanadate [Sigma-Aldrich]). The lysates were sonicated and precleared with protein A agarose 4B beads (Tech & Innovation) for 1 h at 4 °C. Next, the precleared supernatants were subjected to incubation with antibody-conjugated beads for 3 h at 4 °C. The bead-bound proteins and mRNAs were washed using NET-2 buffer, followed by elution with 2× sample buffer (100 mM Tris-HCl [pH 6.8], 10% β-mercaptoethanol, 4% sodium dodecyl sulfate, 15% glycerol, and 0.008% bromophenol blue). The proteins and RNAs bound to the beads were analyzed using western blotting and qRT-PCR, respectively.

Ribosome Profiling

HEK293T cells undepleted or depleted of SRα were harvested with cold phosphate-buffered saline containing 100 μg/mL cycloheximide (Sigma-Aldrich) and lysed in 2 mL of lysis buffer (10 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 100 μg/mL cycloheximide, 1 mM dithiothreitol, 0.2 U/μL RiboLock RNase inhibitor [Thermo Fisher Scientific], and 1× ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail [Roche]), followed by incubation for 10 min on ice. After centrifugation at 10,000g for 10 min at 4 °C, the supernatant was subjected to RNA-IP using either the α-CBP80 or α-eIF4E antibody. The coimmunopurified RNAs were digested with 4 U/μL RNase I (Ambion) for 45 min at room temperature and then isolated with the TRIzol Reagent to obtain ribosome-protected fragments (RPFs). Next, the purified RPFs were subjected to ribosomal RNA depletion using the RiboMinus Eukaryote System v2 (Thermo Fisher Scientific). For the removal of the phosphate groups at the ends of RPFs, the RPFs were incubated with antarctic phosphatase [New England Biolabs (NEB)] for 1 h at 37 °C, followed by heat inactivation at 65 °C for 5 min. The RPFs were treated with T4 polynucleotide kinase (NEB) in 1 mM ATP (adenosine triphosphate; Invitrogen) at 37 °C for 1 h to phosphorylate the 5′ end of RPFs. The size selection of RNAs corresponding to 26 to 32 nucleotides was performed using a 12% tris-borate-EDTA-urea gel system (National Diagnostics). Subsequently, the RNA species were eluted from the sliced gel in 400 μL of 0.3 M NaCl overnight at 4 °C. Next, the eluted RPFs were purified using a Spin-X column (Corning) to remove gel debris and then precipitated with ethanol. Following the manufacturer’s protocol, the library using size-selected RPFs was constructed using TruSeq Small RNA Library Preparation Kits (Illumina).

Ribosome Profiling Analysis

The reads from ribosome profiling and input mRNA-seq were preprocessed in 2 steps: (1) trimming of adapter and poly(A) sequences with Cutadapt (Schibich et al., 2016) and (2) removal of ribosomal sequences with the riboPicker program (Tani et al., 2012) using a customized ribosomal ribonucleic acid library. Reads with lengths greater than 15 bp and Phred quality scores greater than 30 were filtered for the next step. The processed reads were mapped to the reference human genome (hg19) and reference transcriptome using the gene transfer format file from Gencode v19 in STAR aligner software (Sontag et al., 2017). The reads per million values were calculated using the htseq-count Python code (Tajima et al., 1986). Among the variants of the same gene, the longest variant with an reads per million value over one from the input mRNA-seq data was regarded as representative. The coverage of ribosome-protected reads over protein-coding genes was computed using deepTools software (Ramirez et al., 2016). The signal peptides or the first transmembrane domains (TMDs) from the protein-coding genes were predicted using Phobius (Trcek et al., 2013). Only mRNAs with 100% coverage of ribosome-protected reads spanning 20 to 40 amino acids downstream of the first codon of the signal sequence or TMD were considered for metagene plots. For each open reading frame (ORF), the value of each codon was calculated as the relative ratio of the sum of the values. The metagene was represented as the median value of each codon.

Quantification and Statistical Analysis

A 2-tailed equal-variance Student’s t-test was used for statistical analysis. Statistical significance was defined as a P value <.05 or <.01. Data obtained from independent biological replicates are represented as the mean ± standard deviation.

For the heatmap analysis shown in Figure 2E, P values were calculated using Wilcoxon’s signed-rank test.

Fig. 2.

Fig. 2

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NMD of mRNAs encoding a signal peptide depends on the presence of SR. (A) A schematic representation of the NMD reporter mRNAs. Con-RβGl-Norm or -Ter and PPL-RβGl-Norm or -Ter. RLuc, Renilla luciferase; AUG, the translation initiation codon; UAA, the normal translation termination codon; Norm or Ter, respectively, PTC-free or PTC-containing; PPL, the signal peptide sequence derived from preprolactin. (B and C) Effects of downregulation of SRβ, CBP80, or both on the NMD of Con-RβGl or PPL-RβGl mRNAs. HeLa cells either depleted or not depleted of SRβ, CBP80, or both were transiently transfected with the indicated reporter plasmid and phCMV-MUP (a reference plasmid) encoding MUP mRNA. (B) Western blots confirming specific downregulation of the indicated proteins by siRNAs. (C) Relative levels of Con-RβG reporter mRNA (left) and PPL-RβG reporter mRNA (right). The levels of reporter mRNAs were normalized to those of MUP mRNAs. The relative levels of the normalized Norm mRNAs were arbitrarily set to 100%. n = 3; **P < .01. (D) The impact of downregulation of SRβ, CBP80, or both on the half-life of reporter mRNAs. As performed in panel C, except that the cells depleted of the indicated protein were treated with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (a potent transcription inhibitor) before cell harvesting. The levels of the reporter mRNAs normalized to MUP mRNAs at 0 h were arbitrarily set to 100%. The percentages of remaining Con-RβGl mRNAs (left) and PPL-RβGl mRNAs (right) are represented on a log2 scale. Calculated half-life values are depicted in each panel. n = 4. (E) The heatmap of endogenous NMD substrates. Relative amounts of 27 endogenous transcripts, experimentally identified as NMD substrates among the transcripts preferentially enriched in SRP68 IPs, were analyzed using qRT-PCRs. The levels of each NMD substrate were normalized to those of endogenous GAPDH mRNA. The normalized levels of each NMD substrate obtained in undepleted cells were arbitrarily set to 0 on a log2 scale. The heatmap was generated in PermutMatrix (v1.9.3; http://www.atgc-montpellier.fr/permutmatrix/), and P values were calculated using Wilcoxon’s signed-rank test. n = 2; **P < .01; n.s., not significant.

Code Availability

All next-generation sequencing data were deposited in the Sequence Read Archive of the National Center for Biotechnology Information under the accession number GSE 245551.

RESULTS

Translation of the CBC-RNC-SRP Complex is Inhibited at the Transcriptome Level Until it Targets the ER

A previous study based on polysome fractionation experiments with the cytoplasmic extracts of cells expressing reporter mRNA encoding signal peptide revealed that downregulation of SRβ causes a shift of the CBC-RNC-SRP complex containing the reporter mRNA from polysomal to subpolysomal fractions (Park et al., 2021), suggesting that CBC-RNC-SRP is a translationally repressed complex. That is when CBC-RNC-SRP is inefficiently targeted to the ER due to the downregulation of SRβ, the CBC-RNC-SRP remains in a translationally repressed form through the CENTRE.

Based on previous findings obtained using reporter mRNAs, we investigated the effect of CENTRE at the transcriptome level. For this purpose, CBP80-associated and eIF4E-associated mRNPs were immunopurified using α-CBP80 and α-eIF4E antibodies, respectively, from the cells either depleted or not depleted of endogenous SRα using specific siRNAs. Immunopurified mRNPs were subjected to ribosome profiling (Fig. 1A). The specific IPs (Fig. 1B) and downregulation of endogenous SRα (Fig. 1C) were validated using western blotting. The bioinformatics analysis showed that the number of ribosome-protected reads from CBP80-associated mRNAs encoding either a signal peptide or TMD, both of which are known to associate with SRP (Akopian et al., 2013, Chartron et al., 2016, Zhang and Shan, 2014), was significantly increased ∼20 to 40 amino acids downstream of the first amino acid of a signal peptide or TMD when SRα was downregulated (Figs. 1D and F). The increase in the number of ribosome-protected reads after SRα downregulation was not observed in eIF4E-associated mRNAs (Figs. 1E and G). These data indicate that inefficient binding of CBC-RNC-SRP to the SR (caused by SRα downregulation) leads to an elongation arrest of CBC-RNC-SRP, but not eIF4E-RNC-SRP, immediately after a signal peptide or TMD emerges from the exit tunnel of an elongating ribosome. In support of our conclusion, a previous study using reporter mRNA encoding a signal peptide showed that the CBC-RNC-SRP complex preferentially accumulates due to its inefficient binding to SR (Park et al., 2021). Therefore, we clearly demonstrate the impact of CENTRE at the transcriptome level.

Fig. 1.

Fig. 1

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Downregulation of the SRα causes accumulation of the translationally inactive CBC-RNC-SRP complex. (A) Schematic diagram illustrating the experimental approaches for ribosome profiling. IPs were performed with either α-CBP80 or α-eIF4E antibodies and extracts of cells either depleted or not depleted of SRα, to obtain CBP80-associated or eIF4E-associated mRNPs. Subsequently, CBP80-associated or eIF4E-associated mRNPs were subjected to ribosome profiling. (B) Western blots showing specific IPs. (C) Western blots showing specific downregulation of endogenous SRα by siRNA. (D and E) Metagene analysis of ribosome-protected reads from CBC-associated mRNAs (D) and eIF4E-associated mRNAs (E) encoding a signal peptide before and after SRα downregulation. (F and G) Metagene analysis of ribosome-protected reads from CBC-associated mRNAs (F) or eIF4E-associated mRNAs (G) encoding TMD before and after SRα downregulation.

NMD of mRNAs Encoding a Signal Peptide Occurs After Proper Targeting of the mRNA to the ER

NMD occurs primarily during the pioneer translation of newly synthesized CBC-bound mRNA (Ishigaki et al., 2001, Maquat et al., 2010, Ryu and Kim, 2017). Considering that (1) inefficient binding of RNC-SRP to the SR leads to the accumulation of translationally repressed CBC-RNC-SRP (in our study and Park et al. [2021]) and (2) NMD is tightly coupled to a translation event (Ishigaki et al., 2001, Maquat et al., 2010, Ryu and Kim, 2017), it is highly likely that the NMD of mRNAs encoding a signal peptide occurs after proper targeting of RNC-SRP to the ER and resumption of translation elongation.

To test the above possibility, we designed NMD reporter constructs (Fig. 2A), which contain, in sequential order, (1) the sequences either encoding (PPL-RβGl) or not encoding (Con-RβGl) an ER-targeting signal peptide (which interacts with the SRP) derived from PPL (Karamyshev et al., 2014), (2) sequences encoding a renilla luciferase (RLuc), and (3) genomic sequences of β-globin (β-Gl) either lacking (Norm) or containing PTC (Ter). All the tested reporter mRNAs yielded a comparable reduction in the relative ratios of the abundance of a PTC-containing mRNA (Ter) to that of the corresponding PTC-free mRNA (Norm; Fig. S1). In addition, the observed reduction was significantly reversed when UPF1 (a key NMD factor) was downregulated, confirming that all tested reporter mRNAs were efficiently subjected to NMD (Fig. S1).

We investigated the possible role of CENTRE in NMD using NMD reporter mRNAs. First, we observed that the NMD of PPL-RβGl mRNAs significantly diminished after the downregulation of either SRα or SRβ (Fig. S1). In contrast, the NMD of Con-RβGl mRNAs was not significantly affected by the downregulation of either SRα or SRβ, indicating that the NMD of mRNAs encoding a signal peptide preferentially depends on the SR. Second, we observed that the specific downregulation of CBP80, but not SRβ, confirmed using western blotting (Fig. 2B), caused an increase in the relative amount (Fig. 2C, left) and half-life (Fig. 2D, left) of Con-RβGl-Ter mRNAs. Conversely, the downregulation of SRβ, but not CBP80, significantly increased the relative amount (Fig. 2C, right) and half-life (Fig. 2C, right) of PPL-RβGl-Ter mRNAs. Of note, a double knockdown (CBP80 and SRβ) significantly reversed the observed increase. Furthermore, 27 endogenous transcripts, experimentally validated as both NMD substrates (Tani et al., 2012, Yepiskoposyan et al., 2011) and SRP-associating transcripts (Tani et al., 2012), increased in abundance after downregulating either UPF1 or SRα (Fig. 2E). The increase caused by SRα downregulation was significantly reversed by the double knockdown (SRα and CBP80). These data indicate that CENTRE inhibits the NMD of mRNAs encoding a signal peptide until the mRNP is properly targeted to the ER.

NMD of mRNAs Encoding a Signal Peptide Occurs Mostly on eIF4E-associated mRNPs

The observed rescue of the NMD of PPL-RβGl mRNAs by the double downregulation of CBP80 and SRβ (Fig. 2) suggested that the mRNAs encoding a signal peptide are translationally arrested as a form of CBC-associated mRNPs and that their NMD may occur mostly on eIF4E-bound mRNAs only after proper targeting of the CBC-RNC-SRP complex to the ER and subsequent replacement of the CBC by eIF4E. To demonstrate this, we separated CBC-associated mRNPs and eIF4E-associated mRNPs by IPs with the α-CBP80 and α-eIF4E antibodies, respectively (Fig. 3A). Next, the NMD efficiency of the reporter mRNAs was determined by measuring the amount of coimmunoprecipitated reporter mRNAs. Consistent with previous reports showing that NMD predominantly occurs on CBC-associated mRNPs (Ishigaki et al., 2001, Tani et al., 2012), the NMD of CBP80-associated Con-RβGl mRNAs was comparable to that of eIF4E-associated Con-RβGl mRNAs (Fig. 3B). Because CBC-associated mRNPs are precursors of eIF4E-bound mRNPs (Ishigaki et al., 2001, Lejeune et al., 2002), the observed data suggest that the NMD of Con-RβGl mRNAs occurs mostly on CBC-associated mRNPs. In contrast, weak NMD and efficient NMD were observed among CBP80-associated and eIF4E-associated PPL-RβGl mRNAs (Fig. 3B), respectively, indicating that the NMD of mRNAs encoding a signal peptide occurs mostly on eIF4E-bound mRNPs.

Fig. 3.

Fig. 3

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NMD of mRNAs encoding a signal peptide mostly takes place on eIF4E-associated mRNPs. The extracts of HEK293T cells transiently expressing one of the reporter mRNAs and phCMV-MUP (a reference plasmid) were subjected to IPs with either the α-CBP80 or α-eIF4E antibody. Then, coimmunoprecipitated reporter mRNAs were quantitated using qRT-PCRs to measure the efficiency of the NMD. (A) Western blotting to confirm specific IPs. (B) The efficiency of the NMD of each reporter mRNA before or after IPs. The levels of reporter mRNAs before or after IPs were normalized to those of MUP mRNAs. The relative levels of the normalized Norm reporter mRNAs were arbitrarily set to 100%. n = 3; **P < .01; n.s., not significant.

DISCUSSION

Proper targeting of integral membrane proteins and secretory proteins to the ER is ensured by strict quality control pathways (Akopian et al., 2013, Bauer et al., 2015, Karamyshev and Karamysheva, 2018, Pfanner et al., 2019, Zhang and Shan, 2014). Otherwise, improper targeting of integral membrane proteins and secretory proteins may cause misfolding or aggregation of polypeptides, leading to various neurodegenerative pathologies, such as Alzheimer’s and Parkinson’s diseases (Chin et al., 2010, Costa et al., 2018, Hipp et al., 2019, Lakkaraju et al., 2008, Sontag et al., 2017).

CENTRE is a recently characterized molecular process by which the CBC-RNC-SRP complex is maintained in a translationally repressed form until transported to the ER (Park et al., 2021). Efficient CENTRE requires a physical interaction between the CBC and SRP through the N-terminal MIF4G domain of CBP80 and the N-terminal GTPase- domain (NG) domain of SRP54 (Park et al., 2021). In this study, we validate the effects of CENTRE at the transcriptomic level (Fig. 1). In addition, we unravel that the NMD of the newly synthesized mRNA encoding a signal peptide is inhibited by CENTRE and activated only after the CBC-RNC-SRP is properly targeted to the ER (Fig. 2, Fig. 3).

Based on this and previous observations (Park et al., 2021), we propose the following model for NMD at the ER surface (Fig. 4). When the newly synthesized mRNA encodes a signal peptide, the mature mRNA is exported from the nucleus to the cytoplasm with its 5′-cap bound by the CBC and is subject to CBC-driven pioneer translation. The SRP recognized the nascent signal peptide synthesized during pioneer translation. As a result, the RNC-SRP complex is translationally arrested. Concomitantly, the SRP can interact with CBC, stabilizing the CBC-RNC-SRP complex resistant to an IMPβ-mediated replacement of CBC by eIF4E. The resulting complex is maintained as a translationally repressed complex until it is targeted to the ER surface. On the ER surface, SR binds to SRP, disrupting the interaction between the CBC and SRP. Consequently, the CBC-RNC-SRP complex is remodeled as eIF4E-RNC-SRP, and the remaining region of the mRNA is translated. During this process, if the mRNA contains a PTC, the mRNA within the CBC-RNC-SRP complex is not degraded by NMD because the complex is translationally repressed. Only after the mRNA is targeted to the ER and the CBC is replaced by eIF4E, the mRNA is degraded by NMD on the eIF4E-associated mRNPs on the surface of the ER. Thus, CENTRE restricts the NMD of mRNAs encoding a signal peptide on the ER surface.

Fig. 4.

Fig. 4

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Model showing the interplay between CENTRE and NMD. CENTRE translationally represses NMD substrates encoding a signal peptide until the CBC-RNC-SRP is transported to the surface of the ER. After proper targeting, the complex is remodeled to eIF4E-RNC-SRP. Concomitantly, the resumption of translation leads to rapid mRNA degradation by a local NMD. Further details are described in the DISCUSSION section.

When CENTRE is impaired or inefficient, CBC-RNC-SRP is remodeled to eIF4E-RNC-SRP and is promiscuously translated into the cytosol. The resulting polypeptides can accumulate in the cytosol due to a hydrophobic signal peptide and trigger a cytosolic stress response via heat shock factor 1, inducing the expression of heat shock proteins for protein homeostasis (Hipp et al., 2019). Under the conditions where CENTRE is impaired, if the mRNA encoding a signal peptide contains a PTC, the mRNA would be targeted for NMD, as observed in the NMD of mRNAs encoding a soluble protein. Therefore, NMD substrates that encode signal peptides are subject to dual surveillance for tight gene regulation: CENTRE and NMD. When CENTRE is efficient, mRNA would be translationally repressed. In contrast, when CENTRE is inefficient, the mRNA would be degraded by NMD.

The molecular mechanism of the local NMD occurring on the surface of the ER may differ from that of the canonical NMD occurring in the cytosol. A previous report showed that NMD occurring on the surface of the ER is dependent on a peripheral ER membrane protein, neuroblastoma amplified sequence (Anastasaki et al., 2011, Longman et al., 2020, Longman et al., 2007), which functions in the retrograde transport of coat protein complex I vesicles from the Golgi to the ER (Aoki et al., 2009, Civril et al., 2010). On the surface of the ER, neuroblastoma amplified sequence recruits the key NMD factor UPF1 and other NMD factors (SMG5-7), facilitating local NMD (Longman et al., 2020). The detailed molecular mechanisms specific to local NMD need to be addressed in the future.

Author Contributions

M.-K.S., J.-S.W., and Y.K.K. conceived and performed experiments, wrote the manuscript, and secured funding. J.C. and J.P. performed experiments. H.-J.L. provided expertise and feedback.

Declaration of Competing Interests

The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for (MOL CELLS) and was not involved in the editorial review or the decision to publish this article.

Acknowledgments

The authors thank Dr Andrey L. Karamyshev and Dr Philip J. Thomas for providing the plasmids expressing PPL. This work was supported by a National Research Foundation (NRF) of Korea grant funded by the Korean government (Ministry of Science, ICT and Future Planning; NRF-2015R1A3A2033665 and NRF-2018R1A5A1024261).

Footnotes

Appendix A

Supplemental material associated with this article can be found online at: doi:10.1016/j.mocell.2024.100049.

ORCID

Min-Kyung Shin https://orcid.org/0009-0009-9335-5137

Jeeyoon Chang https://orcid.org/0000-0002-0117-9116

Joori Park https://orcid.org/0000-0003-2624-5164

Hyuk-Joon Lee https://orcid.org/0000-0001-7568-2307

Jae-Sung Woo https://orcid.org/0000-0001-9163-3433

Yoon Ki Kim https://orcid.org/0000-0003-1303-072X

Appendix A. Supplemental material

Fig. S1

NMD efficiently degrades reporter mRNAs encoding a signal peptide in an SR-dependent manner. HeLa cells depleted or not depleted of UPF1, SRα, and SRβ were transiently transfected with the indicated NMD reporter plasmid and phCMV-MUP (a reference plasmid) encoding MUP mRNA as a control. (A) Western blots confirming specific downregulation of the indicated proteins by siRNAs. (B) Relative levels of Con-RβG reporter mRNA (left) and PPL-RβG reporter mRNA (right). The levels of reporter mRNAs were normalized to those of MUP mRNAs. The relative levels of the normalized Norm mRNAs were arbitrarily set to 100%. n = 4; **P < .01.

mmc1.pdf (92.9KB, pdf)

.

Table S1

Oligonucleotides used for qRT-PCRs

mmc2.xlsx (12KB, xlsx)

.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1

NMD efficiently degrades reporter mRNAs encoding a signal peptide in an SR-dependent manner. HeLa cells depleted or not depleted of UPF1, SRα, and SRβ were transiently transfected with the indicated NMD reporter plasmid and phCMV-MUP (a reference plasmid) encoding MUP mRNA as a control. (A) Western blots confirming specific downregulation of the indicated proteins by siRNAs. (B) Relative levels of Con-RβG reporter mRNA (left) and PPL-RβG reporter mRNA (right). The levels of reporter mRNAs were normalized to those of MUP mRNAs. The relative levels of the normalized Norm mRNAs were arbitrarily set to 100%. n = 4; **P < .01.

mmc1.pdf (92.9KB, pdf)
Table S1

Oligonucleotides used for qRT-PCRs

mmc2.xlsx (12KB, xlsx)

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