The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT–quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND–NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.

NCBP2/NCBP3 curtailed NCBP1 association with 5Ј-caps demonstrated that NCBP1 alternatively forms CBC with NCBP2 or NCBP3 (9). Further evidence demonstrated that NCBP3 is essential to mount a precise and appropriate stress response (34). NCBP3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge, suggesting that NCBP3 supports expression of stressresponsive genes (34). Given that NCBP2 and NCBP3 function redundantly to support nuclear processing of mRNAs and that NCBP3-deficient cells evoke a reduced antiviral response, we posited that the noncanonical CBC (consisting of NCBP1/ NCBP3) is important for translation of stress response genes (31). The gap in knowledge remains in determining the involvement of the noncanonical CBC in translation of select mRNAs.
AP-1 transcription factors are stress-response proteins (JUND, JUNB, c-Jun) (35). JUND, a negative regulator of cell proliferation, is among the subset of host mRNAs selectively translated during post-translational down-regulation of eIF4E (19). Previous investigations identified translation of JUND, and retroviral transcripts depend upon nuclear RNA helicase A (DHX9/RHA) (36). DHX9/RHA binds to a specific class of post-transcriptional control element (PCE) in JUND and several retroviral 5Ј-UTRs to stimulate polysome formation (36 -38). RHA down-regulation or ectopic expression of the two N-terminal RNA-binding domains (RBDs) were found to attenuate the synthesis of JUND and HIV-1 virion proteins (36,38). The RHA co-factors necessary for assembling polysomes remain to be identified. Given the mutual engagement of NCBP1 and RHA to select mRNAs, we posited that NCBP1 and RHA are components of the same RNP that facilitates cap-dependent translation of JUND.
Antiserum to NCBP2 or NCBP3 independently co-precipitated NCBP1 (Fig. 1A), corroborating the previous finding that NCBP1 independently enriches NCBP2-or NCBP3-bound 5Ј-capped mRNAs (9). The RHA immune complexes co-precipitated NCBP1 and NCBP2 or NCBP3, and the results of seven replicate experiments confirmed the lack of detectable eIF4E and eIF4G, recapitulating different mRNPs. Reciprocal IPs validated each of the indicated interactions (Fig. 1A). Reciprocal co-IP of selected proteins from HEK293 lysates with specific antisera are shown (bold type). Bound proteins were eluted and analyzed by WB with the antisera indicated on the right. Isotype-specific IgG served as the negative control, and input cell lysate served as the positive control. The results are summarized in the tables below each WB. A, IP of NCBP1, RHA, NCBP2, or NCBP3. B, IP of eIF4E, eIF4G, or DDX3. The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained molecular mass markers (lanes M). The same image of the molecular mass markers was used for each panel. ϩ, positive co-IP. *, nonspecific background. The WBs were subjected to ImageJ densitometry quantification (Table S1).

Non-eIF4E translation is supported by nuclear protein
NCBP3 co-precipitated DDX3, but not other components of the eIF4E RNPs. The observed NCBP3-DDX3 interaction may be attributable to DDX3 in transitional interaction between NCBP-bound and eIF4E-bound 5Ј-caps. In summary, the immune complexes of NCBP1-RHA did not bind DDX3, whereas eIF4E did not bind RHA. The results suggest that there are multiple NCBP3 complexes.
To ascertain whether NCBP1-RHA RNP is conserved in other cell types, we investigated cyto lysates of several other cell lines such as simian fibroblasts (COS), HTLV-1-transformed SLB-1 human T lymphocytes, and CEMϫ174 human T lymphocytes. The studies assessed co-precipitation of NCBP1 and RHA with or without NCBP2, NCBP3, or eIF4E. The results from three independent experiments demonstrated that NCBP1-RHA complexes selectively interact with NCBP3 or NCBP2 and are mutually exclusive of eIF4E in these cell lines (Fig. 2, A and B). These findings were not surprising because prior studies have demonstrated NCBP1, NCBP3, or NCBP2 and RHA exhibit high-level amino acid sequence conservation across Mammalia and translation of viral mRNAs requires RHA of bovine, feline, human, and simian origin (36).

NCBP1-NCBP3-RHA mRNPs exclusive of NCBP2 are components of JUND polysomes
Next, experiments employed tandem IPs to document whether RHA and NCBP1, NCBP3, or NCBP2 are components of the same RNP in polysomes (Fig. 3, A and B). Exogenous expression of pFLAG-RHA was performed, and the HEK293 cells were collected in the lysis buffer and designated as Input cyto lysate (Fig. 3B). Next the Inputs were subjected to sucrose gradient centrifugation, and polysome fractions were collected (Fig. 3A) and subjected to WB (Fig. 3C). WB by several antiserum verified FLAG-RHA, NCBP3, NCBP1, and NCBP2 in the Input cyto lysate (Fig. 3C, lane 1). Noticeably, FLAG-RHA, NCBP3, and NCBP1 were enriched in polysomes, whereas NCBP2 was absent (Fig. 3C, lane 2). These results exposed functional differences between canonical CBC (NCBP1-NCBP2) and noncanonical CBC (NCBP1-NCBP3). The lack of NCBP2 detection in polysomes corroborated previous demonstration of CBC activity in pioneer round, but not steady-state translation (41).
Next, the Input and polysome samples were incubated with FLAG antiserum conjugated to protein G magnetic beads. Immune complexes were washed and collected by competitive elution with 3ϫ FLAG peptides (250 g/ml) as depicted in Fig.  3B. The WB of the cyto IP verified the enrichment of FLAG-RHA and the co-precipitation of NCBP1, NCBP2, and NCBP3 (Fig. 3C, lane 3).
Moreover, the WB of the polysome IP verified the enrichment of FLAG-RHA and the co-precipitation of NCBP1 and NCBP3. Importantly, NCBP2 was not detectable in the polysome IP (Fig. 3C, lane 4), corroborating the lack of NCBP2 in the WB of the collected polysomes (Fig. 3C, lane 2). There was no detectable enrichment of the proteins in the protein G-negative control (Fig. 3, lane 5). Next, the eluates were subjected to tandem IP with NCBP1 antiserum conjugated to protein G magnetic beads and analyzed by WB (Fig. 3B). The results validated that the NCBP1 tandem IP captured FLAG-RHA from cyto and polysome samples (Fig. 3C, lanes 6 and 7, FLAG-RHA panel). The tandem IP ascertained NCBP3 and NCBP2 enrichment in the cyto tandem IP (Fig. 3C, lane 6). However, the polysome tandem IP captured NCBP3 but failed to capture NCBP2 (Fig. 3C, lane 7). There was no enrichment of these proteins in the IgG isotype control (Fig. 3C, lanes 8 and 9). We concluded that NCBP3 is the mutual component of FLAG-RHA-NCBP1 RNPs in polysomes. , or isotype IgG from lysates of HEK293, COS7, SLB-1, or CEMϫ174 cells depleted of nuclei. Immune complexes (bold type) were washed, collected in SDS sample buffer, and analyzed by WB with the antisera indicated on the right. In each case, the input cell lysate served as the positive control, and the isotype-specific IgG served as the negative control for background immunoreactivity. The results are summarized in the table below each WB. The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained molecular mass markers (M). The same image of the molecular mass markers was used for each panel. ϩ, positive co-IP. *, nonspecific background. The WBs were subjected to ImageJ densitometry quantification (Table S1).

Non-eIF4E translation is supported by nuclear protein
To validate that JUND is a component of the FLAG-RHA-NCBP1-NCBP3 RNPs, replicate tandem IP samples were extracted with TRIzol-LS, and the co-precipitating RNA was collected and subjected to RT-qPCR using JUND and GAPDH specific primer pairs. The JUND transcripts were readily detectable in the FLAG-RHA-NCBP1-NCBP3 RNP, whereas GAPDH transcripts were less than the minimum detectable (ϽMD) (Fig. 3D). Both JUND and GAPDH were ϽMD in the IgG controls (Fig. 3D). These results validated that FLAG-RHA-NCBP1-NCBP3 are mutual components of JUND polysomes.

RHA tethers NCBP1-NCBP3 to polysomes through RNA
NCBP1-NCBP3 may be tethered to RHA through RNA or via protein-protein interaction, which would be sensitive or resistant to RNase treatment, respectively. To examine the nature of the interaction between RHA and NCBP1-NCBP3, reciprocal IPs were performed using cyto lysates treated with RNase A. The cyto lysate was incubated with RHA or NCBP1 antiserum conjugated to protein G magnetic beads with or without RNase A (1 g/ml) for 2 h. Immune complexes were washed and collected in SDS sample buffer. The RHA IP and NCBP1 IP co-precipitated RHA, NCBP1, NCPB3, and NCBP2 ( Fig. 4A, lanes 1 and 3). However, RNase A treatment of the RHA IP eliminated NCBP1, NCBP3, and NCBP2 (Fig. 4A, lane 2). RNase A treatment of the NCBP1 IP eliminated RHA co-precipitation, establishing that the interaction is RNA-dependent (Fig. 4A, lane 4). We concluded NCBP1 is tethered to RHA through RNA.
Because RHA binding to PCE RNA requires the N-terminal double-stranded (ds) RBDs and is eliminated by mutation of surface-exposed lysine residues (K54A, K55A, and K236E) (42) (Fig. 4B), we postulated that the same residues are critical for RHA interaction with NCBP1-NCBP3 or NCBP1-NCBP2. To examine this, HEK293 cells were transfected with FLAG-RHA-WT or FLAG-RHA modified by the following substitutions: K54A,K55A (KK), K236E, or K54A,K55A,K236E (KKK) and subjected to reciprocal IPs using antiserum to FLAG or NCBP1.
Reciprocal IP was performed using antiserum to NCBP1. NCBP1 co-precipitated FLAG-WT, KK or K236E, but not KKK (Fig. 4D). NCBP3 and NCBP2 interactions were maintained. The co-precipitation of ribosomal proteins rpS6 and rpL5 by FLAG or NCBP1 was diminished but not abrogated by the lysine mutations (Fig. 4, C and D). The results demonstrated that NCBP1-NCBP3 and NCBP1-NCBP2 are tethered through RNA to the N-terminal dsRBDs of RHA.

RHA is necessary for the assembly of NCBP1-NCBP3-JUND polysomes and mTOR-resistant JUND translation
Next, we sought to establish whether RHA-PCE interaction is necessary for the assembly of NCBP1-NCBP3 polysomes. HEK293 cells were transfected with siRNAs targeting NCBP3 or RHA or with nontargeting (NT) control siRNA. 24 h posttransfection, the cell lysates were analyzed by WB, and total Tandem IPs were employed to isolate components of the same RNP from HEK293 cells transfected with pFLAG-RHA. Input cyto lysates were subjected to sucrose gradient centrifugation, and polysomes were collected. A, A 254 spectrometry (red line) of sucrose gradient. B, outline of tandem IP. The polysome fractions were combined, precipitated, and resuspended in low-salt buffer (Input poly). Aliquots of the cyto (white tube) and polysome samples (blue tube) were incubated with FLAG antiserum conjugated to protein G beads. The beads were washed and incubated with 3ϫ FLAG peptides to elute RNPs. A fraction of the eluate was reserved for WB, and the remaining eluate was incubated with NCBP1 antiserum conjugated to protein G beads. The beads were extracted in SDS buffer for WB analysis or with TRIzol to isolate RNA for subsequent RT-qPCR with gene-specific primers. C, WB of Input cyto and polysomes (lanes 1 and 2), eluates of FLAG IP (lanes 3 and 4), eluate of Protein G IP (control for FLAG IP, lane 5), eluates of NCBP1 IP (lanes 6 and 7), and eluates of IgG IP (control for NCBP1 IP; lanes 8 and 9). The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained molecular mass markers (M). The same image of the molecular mass markers was used for each panel. *, nonspecific band. D, JUND and GAPDH copies by RT-qPCR. The results represent the means of three independent experiments with standard deviation.

Non-eIF4E translation is supported by nuclear protein
cellular RNA was analyzed by RT-qPCR using gene-specific primers. WB showed that NCBP3 and RHA proteins were down-regulated by the specific siRNA, whereas NCBP1 and GAPDH protein expression was unaffected (Fig. 5A). The RT-qPCR recapitulated the down-regulation of ncbp3 and dhx9/ rha by the specific siRNAs relative to GAPDH (Fig. 5B). The cyto lysates were subjected to sucrose density sedimentation and A 254 spectrometry to identify the rRNA profile. The results of three independent experiments identified that the magnitude of polysomes was modestly diminished by RHA down-regulation compared with the siNT control (Fig. 5C). The siNCBP3 treatment did not diminish polysomes and reproducibly increased the robustness of the 60S and 80S peaks (Fig. 5C). The results indicate that NCBP3 has a generalized effect on ribosome recruitment.
The distribution of mRNAs in the gradient fractions was determined by RT-qPCR. The down-regulation of RHA significantly reduced JUND polysomes, whereas no change was observed in GAPDH polysomes (Fig. 5D). The results recapitulated previous metabolic labeling assays showing that RHA down-regulation reduces the synthesis of JUND, but not GAPDH protein (36). The down-regulation of NCBP3 significantly increased PIC (48S and 60S mRNPs) and diminished polysomes (Fig. 5D). The JUND PIC increased from 2% to 15%, whereas heavy polysomes decreased from 69% to 58% of the RNA copies, accounting for the redistribution to PIC. The GAPDH PIC increased from 12% to 22%, and light polysomes decreased from 20% to 10% of the RNA copies, accounting for the redistribution in RNA copies. The increase in PIC was 4-fold greater for JUND than GAPDH polysomes (Fig. 5D). The results do not exclude the possibility of redundant activity by RHA-NCBP1-NCBP2 as a result of NCBP3 down-regulation. Indeed, NCBP2 and NCBP3 were shown to function redundantly to support nuclear processing of mRNAs (9). We concluded that RHA-NCBP1-NCBP3 activity is important for JUND polysome assembly at steady state. The prospect of RHA-NCBP1-NCBP3 RNP activity during mTOR down-regulation was an important open issue. Therefore, we evaluated the effect of mTOR down-regulation on the assembly of JUND polysome components, RHA-NCBP1-NCBP3. The small molecule mTOR inhibitor, Torin-1 activates the allosteric inhibitor of eIF4E, hypophosphorylated 4E-BP1. Torin-1 titration experiments were first carried out to establish treatment time and optimal dose to activate hypophosphorylated 4E-BP1 while maintaining ribosome profiles and no change in cell viability. The titration experiments identified cells exposed to 50 nM Torin-1 for 18 h maintained consistent polysome profiles and up-regulated hypophosphorylated 4E-BP1 in several replicate experiments with the cell viability remaining similar to mock-treated cells. Immunoblotting of the cell lysates documented that RHA, NCBP1, NCBP3, and other proteins maintained steady state at 50 nM Torin-1 for 18 h (Fig. 6A and Fig. S2).
To evaluate the capacity for cells to recover from the Torin-1 treatment, the medium was exchanged, and cells were cultured without Torin-1 for 1 or 18 h. Immunoblotting validated recovery of hyperphosphorylated 4E-BP1 in the cells subject to shortterm (1 h) and long-term (18 h) culture (Fig. S3). We concluded the treatment conditions employed were appropriate to maintain polysome profiles and cell viability for 18 h.
Next, HEK293 cells were transfected with RHA-specific or NT siRNAs (mock) for 24 h and then exposed to 50 nM Torin-1 for 18 h. The WB on cyto lysates validated that RHA was downregulated by siRNA (Fig. 6B, lanes 2 and 4), and hypophosphorylated 4E-BP1 was up-regulated by Torin-1 treatment (Fig. 6B,  lanes 3 and 4). Tubulin was unaffected by the treatments. The results identified JUND proteins reduced in response to the RHA down-regulation (Fig. 6B, compare lanes 1, 2, and 4) but not by Torin-1 treatment (compare lanes 1 and 3). Conversely, GAPDH protein was reduced by Torin-1 but not by RHA  (Table S1).

Non-eIF4E translation is supported by nuclear protein
down-regulation. The results verified JUND translation is resistant to mTOR inhibition.
The cyto lysates were subjected to sucrose density sedimentation and A 254 spectrometry. As expected, ribosomal profiles were diminished by the Torin-1 treatment (Fig. 6C). Next, RT-qPCR was performed on the input lysate, and the RNA was isolated from the gradient fractions. The results verified that RHA down-regulation significantly reduces JUND polysomes (Fig. 6D, upper panel) but not GAPDH polysomes (Fig. 6D,  lower panel). Torin-1 treatment significantly down-regulated GAPDH polysomes (Fig. 6D, lower panel) but not JUND polysomes (Fig. 6D, upper panel). As expected, the combination of Torin-1 treatment and RHA down-regulation significantly reduced both JUND and GAPDH polysomes (Fig. 6D). The distribution across the sucrose gradients of RHA, NCBP1, NCBP3, and other proteins was evaluated by immunoblotting (Fig. S4). The results validated that Torin-1 eliminated eIF4E from polysome fractions, whereas RHA, NCBP3, and NCBP1 remained.
Next, replicate polysome fractions were combined, and proteins were co-precipitated by NCBP1 or eIF4E antiserum. The immunoblots validated NCBP1 co-precipitated RHA and NCBP3 polysomes in the presence or absence of the Torin-1 treatment, whereas neither NCBP2 nor eIF4E were detectable (Fig. 6E). The eIF4E immunoprecipitates enriched eIF4G, and these polysomes were eliminated by Torin-1 (Fig. 6F). None of the candidate proteins were detected in the IgG isotype controls (Fig. 6, E and F).
The IP samples were extracted with TRIzol-LS, and the coprecipitating RNAs were collected and subjected to the RT-qPCR. In results of three independent experiments, JUND transcripts were readily detectable in immunoprecipitated NCBP1 polysomes, whereas GAPDH transcripts were not (ϽMD) (Fig.  6E, below WB). In contrast, the eIF4E polysomes enriched GAPDH transcripts, but not JUND (ϽMD) (Fig. 6F, below WB). The IgG IPs did not enrich either JUND or GAPDH (ϽMD) (Fig. 6, E and F). These findings demonstrated the assembly of the JUND polysome components, RHA-NCBP1-NCBP3, is mTOR-independent.

Discussion
This study has identified a specialized translation pathway for cap-dependent translation of JUND that does not utilize eIF4E or eIF4G nor NCBP2. Strong evidence is provided that  NCBP1 and RHA are components of the same RNP that is facilitating cap-dependent translation of JUND. Moreover, JUND polysomes are composed of RHA-NCBP1-NCBP3 and assemble at steady state and during mTOR inhibition. Current evidence indicates that the RHA-NCBP1-NCBP3 cap-dependent translation mRNP is not regulated by eIF4E levels and appears to be a constitutively active pathway rather than a rescue pathway.

Non-eIF4E translation is supported by nuclear protein
In addition to JUND, several retroviruses contain the RHAresponsive element; PCE and RHA-NCBP1-NCBP3 are likely involved in the translation control of these mRNAs. PCE does not support internal ribosome entry, and PCE translation activity has been shown to be cap-dependent (37). Prior research established the RHA-PCE RNA interaction neutralizes structural barriers within the 5Ј-UTR that repress ribosome scanning to promote efficient translation (36). Our new findings provide strong evidence that RHA is necessary for the assembly of JUND polysomes at steady state and during mTOR inhibition (Fig. 7). Moreover, NCBP1-NCBP3 and NCBP1-NCBP2 are tethered to RHA through JUND by the N-terminal dsRBDs of Statistical significance is indicated with asterisks: *, p Յ 0.05; **, p Յ 0.005. E and F, the light and heavy polysome fractions were combined, and proteins were precipitated and subjected to IP with NCBP1 or eIF4E antiserum. Co-precipitates were subjected to WB with the indicated antiserum or RNA extraction followed by RT-qPCR. Isotype-specific IgG were used as negative controls. The antiserum detected the specific proteins on the immunoblots, as shown relative to the prestained molecular mass markers (M). The same image of the molecular mass markers was used for each panel. *, nonspecific band. The table below each WB panel shows the number of copies of JUND and GAPDH relative to IgG control detected by RT-qPCR.

Non-eIF4E translation is supported by nuclear protein
RHA. The molecular basis by which RHA-PCE interaction subverts canonical exchange of the nuclear cap-binding proteins to eIF4E remains an open issue.
JUND is a member of the Jun family of transcription factors that dimerize with c-Jun, Fos, or other family members to form AP-1 (29,35). Recently, the 5Ј-UTR of c-jun was shown to assemble non-eIF4E translation initiation complexes (29). In preliminary studies, we observed c-jun RNA copies are enriched in RHA-NCBP1-NCBP3 immune complexes (5.3 ϫ 10 4 and 3.1 ϫ 10 4 , minus and plus Torin-1, respectively) at levels similar to JUND in Fig. 6E. Next, we replicated the tandem IP experiment and identified the enrichment of c-jun-RHA-NCBP1 mRNPs in cyto and polysomes (11 ϫ 10 4 and 39 ϫ 10 4 copies). Further experimentation is warranted to establish that the c-jun 5Ј-UTR recapitulates activity of JUND PCE and possibly retroviral PCEs, which is necessary for RHA interaction (36,(43)(44)(45) and mTOR-resistant translation.
AP-1 is a critical regulator of nuclear gene expression during T-cell activation, innate response to viral infections, and antitumor immune response through type I interferons and proinflammatory cytokines (46). Dysregulation of AP-1 is hallmark of viral pathogenesis, neoplastic transformation, and tumor progression (35,46). Likewise, dysregulation of dhx9/rha is associated with productive viral infection (31,37,38,45,(47)(48)(49)(50) and tumor survival (51)(52)(53)(54). The recent finding that NCBP3 is essential to mount a precise antiviral response (34) posits dysfunction in a RHA-NCBP1-NCBP3 translation pathway that contributes to deficient innate response. The DHX9, DExHbox helicase 9 (alias RNA helicase A) gene is within the 1q25 prostate/lung/breast cancer locus, and gene mutation or overexpression is pervasive in human clinical samples (55). We speculate that mutations in PCE or RHA residues identified in this report provide biomarkers for neoplastic transformation.

Cell culture and transfection
Dulbecco's modified Eagle's medium or RMPI culture medium were supplemented with 10% fetal bovine serum and 1ϫ antibiotic-antimycotic solution (Gibco) and used to culture HEK293 and COS7 cells or SLB-1 and CEMϫ174 cells, respectively. The cell lines were obtained from ATCC, and low-passage cultures were used in the experiments. In addition, mycoplasma testing was performed periodically and on a weekly basis. The cells were authenticated and documented free of mycoplasma based on morphological evaluation of cells plated at high and low culture densities under a microscope, and the cell morphology images were maintained for comparisons.
As described previously, cDNA was generated using Omniscript (Qiagen), random primers (Invitrogen), cellular RNA (2 g), co-precipitated RNA samples (56), or tandem IP samples (15,57), followed by RT-qPCR using gene-specific primers, which are provided in Table S4. All experiments were performed in three or more biological replicates.  Right, canonical engagement of NCBP1-NCBP2 at the 5Ј-cap is depicted by mRNA lacking PCE. Consequential to nuclear export, CBC undergoes exchange to eIF4E, which engages eIF4G and other factors to assemble polysomes. In common, both mRNPs experience the exchange of PABPN to PABPC1. The eIF4E-eIF4G-dependent polysome assembly is abrogated upon the inhibition of mTOR because of down-regulated eIF4E activity.

Non-eIF4E translation is supported by nuclear protein Density sedimentation and analysis of RNPs
Published protocols were used to perform density sedimentation and A 254 spectrometry (58). Briefly, the cells were transfected for 24 h and treated with Torin-1 (50 nM) for 18 h. The culture medium was supplemented with 0.1 mg/ml cycloheximide (CHX) for 10 min. The cells were washed twice with icecold 1ϫ PBS containing 0.1 mg/ml CHX and collected in 5 ml of ice-cold 1ϫ PBS with CHX by scraping. The cells were subjected to centrifugation at 1500 rpm for 4 min at 4°C and resuspended in 0.75 ml of low salt buffer (20 mM Tris-HCl, pH 7.5, 3 mM MgCl 2 , 10 mM NaCl, 2 mM DTT, 1ϫ protease inhibitor mixture EDTA-free, 5 l/ml RNase Out) and allowed to swell on ice for 5 min. The cells were lysed on ice by the addition of 0.25 ml of lysis buffer (0.2 M sucrose, 0.5% Nonidet P-40, and 0.1% Triton X-100 prepared in low salt buffer) and 10 strokes in a Dounce homogenizer (Kimble Chase). The lysates were depleted of nuclei by centrifugation at 13,000 rpm for 1 min at 4°C to collect cytoplasm. The lysates were used for the sucrose gradients and IPs. Equivalent amounts of RNA OD units across samples were layered on the top of 10 -50% sucrose prepared in 10 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 100 mM KCl, 2 mM DTT, 0.1 mg/ml CHX and centrifuged using a SW41 rotor (Beckman Coulter) for 2 h 40 min at 35,000 rpm at 4°C. Polysome profiles were generated by continuous monitoring of RNA absorbance at 254 nm by the ISCO UA-6 absorbance detector unit (Brandel) and fractionated into 22 equivalent volume (0.5 ml) fractions by the ISCO Foxy R1 fraction collector. Brandel Peak Trace software was used to generate the corresponding profile traces. The proteins were precipitated from the collected fractions by ProteoExtract protein precipitation kit (EMD Millipore) according to the manufacturer's instructions or by TCA precipitation. The fractions were supplemented with ice-cold TCA (final concentration, 20%), vortexed, and incubated overnight at Ϫ20°C. The samples were centrifuged at 12,000 rpm for 15 min at room temperature, and the pellets were washed twice with three volumes of ice-cold acetone. The precipitated proteins were resuspended in equivalent volume of low salt buffer and used for WB or tandem IP. The even fractions were used for protein precipitation, and the odd fractions were used for RNA extraction in TRIzol-LS (Ambion).

IP and tandem IP
Dynabeads Protein G (30 l) (Invitrogen) were washed two times in 10 bed volumes of IP lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40). The beads were incubated with the antiserum in 10 bed volumes of IP lysis buffer containing 1 mM BSA for 45 min at room temperature. The antibodies used for IP are listed in Table S2. The beadantibody complexes were washed once in 10 bed volumes of IP wash buffer (20 mM Tris-HCl, pH 7.4, 300 mM NaCl, 0.5% Nonidet P-40) and incubated with 300 g of cell lysate at 4°C for 2 h with rotation. The enriched immune complexes were washed four times in IP wash buffer and collected by boiling with 1ϫ SDS sample buffer.
Tandem IP was performed on the cyto lysates from pFLAG-RHA-transfected HEK293 cell cultured in three 15-cm plates.
Dynabeads Protein G (100 l) (Invitrogen) was washed twice in 10 bed volumes of lysis buffer (20 mM Tris-HCl, pH 7.5, 3 mM MgCl 2 , 10 mM NaCl, 2 mM DTT, 1ϫ protease inhibitor mixture EDTA-free, 5 l/ml RNase Out, 0.2 M sucrose, 0.5% Nonidet P-40, and 0.1% Triton X-100) and incubated with 10 l of FLAG antiserum (1 mg/ml; Sigma) in 10 bed volumes of lysis buffer containing 1 mM BSA for 45 min at room temperature. 500 g of cyto lysate was diluted to 1 ml in the lysis buffer, and the final concentration of NaCl was adjusted to 300 mM. The diluted lysate was mixed with protein G-anti-FLAG complex and incubated at 4°C for 2 h. The RNPs captured by the anti-FLAGconjugated protein G beads were subsequently washed twice (1 ml each) in ice-cold wash buffer I (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% Nonidet P-40) followed by two washes in icecold wash buffer II (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40). The samples were incubated with one bed volume of wash buffer II containing 250 g/ml 3ϫ FLAG peptide and 5 l/ml RNase Out with gentle shaking at 4°C overnight. The suspension was cleared by magnetic force followed by centrifugation at 12,000 rpm for 5 min at 4°C. The cleared suspension was incubated with protein G (50 l)-anti-NCBP1 (4 l) complex for 2 h at 4°C. The captured RNP complexes were washed four times in wash buffer II and divided into two equal parts for WB and RNA isolation. The proteins precipitated from polysome fractions were processed in a similar manner. 10% of each sample volume was reserved from each step of the tandem IP for WB. The isolated proteins were resolved using commercial 4 -15% gradient SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membrane and immunoblotted.

Statistical data analysis
Three or more independent experiments were performed for each assay and results were combined to define the means Ϯ S.D. Statistical significance was assessed using a two-tailed Student's t test, and a p value (*) of Ͻ 0.05 was considered to be significant. Bar graphs present mean and S.D. of three independent experiments, and the precise values from the individual experiments are denoted by small dots. Statistical significance is denoted with asterisks: *, p Յ 0.05; **, p Յ 0.005.

Data availability
The raw RNA copies from the polysome analysis are available from the corresponding author upon request. All other data are contained within the manuscript.