SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, which up-regulate the translation of target mRNA and have been studied in a wide range of applications for biological and therapeutic tools, although the molecular mechanism is unclear. Here, we focused on the kinetic distribution of target mRNAs and SINEUP RNAs by performing co-transfection of expression vectors for these transcripts into human embryonic normal kidney cells (HEK293T/17) to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was one of the key phenomena. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms on the fate of regulatory RNAs implicated in efficient protein translation.


Introduction
More than 70% of the mammalian genome is transcribed into RNAs The ENCODE Project Consortium, 2012). The majority of these RNAs do not encode proteins and are called non-coding RNAs. A large fraction of non-coding RNAs are found as sense-antisense pairwise transcripts that are co-regulated at the transcription level . Although some of these non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), have been intensely studied, the biological function of 98.5% of the long non-coding RNAs (lncRNAs), which are defined as non-coding RNAs longer than 200 nucleotides (Kapranov et al, 2007), are still unknown (de Hoon et al, 2015). Importantly, some lncRNAs have regulatory functions at the transcriptional or translational levels (Hu et al, 2012;Rinn & Chang, 2012;Quinn & Chang, 2016).
We previously found AS-Uchl1, a natural antisense (AS) lncRNA, was able to enhance the translation of its sense Uchl1 (ubiquitin carboxyterminal hydrolase L1) mRNA in mouse dopaminergic cells (Carrieri et al 2012). The functional element embedded in AS-Uchl1 is an inverted SINEB2 (Short Interspersed Nuclear Element B2) that acts as an effector domain (ED) and is essential for up-regulating protein synthesis (Zucchelli et al, 2015). This element enhanced the UCHL1 protein level without changing the Uchl1 mRNA level. In mouse dopaminergic neurons, mature Uchl1 mRNA is predominantly localized in the cytoplasm, whereas the natural AS-Uchl1 RNA is localized in the nucleus at steady state levels. Interestingly, in rapamycin-induced stress conditions, endogenous AS-Uchl1 RNA is transported to the cytoplasm to increase Uchl1 mRNA association to heavy polysomes and enhances its translation (Carrieri et al, 2012).
We also found that other natural antisense lncRNAs containing SINEs specifically up-regulate translation of the overlapping sense mRNAs, and termed such functional class of lncRNAs "SINEUPs" (Zucchelli et al, 2015). In addition to the inverted SINEB2, SINEUPs also require an overlapping region known as the target binding domain (BD) of the sense mRNA (Zucchelli et al, 2015). We used the configuration of the original natural SINEUPs to produce a synthetic SINEUP targeting green fluorescent protein (SINEUP-GFP) by replacing the BD of AS-Uchl1 with that of the target enhanced green fluorescent protein (EGFP) mRNA. SINEUP-GFP successfully up-regulated the translation of EGFP mRNA in an ED-dependent fashion maintaining target mRNA levels unchanged. Despite the functional evidence, the mechanism by which natural and synthetic SINEUPs enhance the translation of a target mRNA is still unknown; in particular, the fate of SINEUP RNAs after transcription, such as their subcellular distribution and contribution to protein translation, is unclear.
Here we show that SINEUP RNA interacts with nucleocytoplasmic proteins such as PTBP1 (polypyrimidine tract binding protein-1) and HNRNPK (heterogeneous nuclear ribonucleoprotein K) that are essential for RNA localization and translational initiation assembly.

SINEUP-GFP enhances EGFP mRNA translation
To confirm translational up-regulation activity by SINEUP constructs, we produced synthetic SINEUPs targeting EGFP mRNA ( Fig 1A). We previously reported that SINEUP-GFP enhances EGFP levels (Indrieri et al, 2016) more efficiently when cloned into the pCS2+ plasmid than when cloned into pcDNA3.1 plasmid as shown in our previous studies (Carrieri et al, 2012, Zucchelli et al, 2015. The expression levels of RNAs produced by different plasmids often differ due to different promoters, stability, and polyadenylation status, and in the current study we found that the level of SINEUP RNA transcripts was higher for pCS2+ than for pcDNA3.1 ( Fig EV1). Therefore, pCS2+ was used for all subsequent experiments. We then examined the EGFP up-regulation activities of SINEUP-GFP and of the deletion mutants constructed in this study; the BD mutant (SINEUP-SCR), the ED deletion mutant (SINEUP-ΔSB2), and the Alu element deletion mutant (SINEUP-ΔAlu) in pCS2+ ( Fig 1A). Consistent with previous studies, SINEUP-GFP in pCS2+ showed approximately 2-fold induction of EGFP level compared to the No-insert control (vector containing no SINEUP construct) (Fig 1B, C). SINEUP-SCR and SINEUP-ΔSB2 did not significantly elevate the EGFP level, but the Alu element deletion mutant (SINEUP-ΔAlu) and SINEUP-GFP did significantly elevate the EGFP level. Because none of the constructs significantly affected the EGFP mRNA level (Fig 1D), the results indicate that translation of EGFP mRNA was induced by SINEUP-GFP and SINEUP-ΔAlu, but not by SINEUP-SCR or SINEUP-ΔSB2.

SINEUP RNAs co-localized with EGFP mRNAs in the cytoplasm
We previously showed that, in mouse dopaminergic neurons, mature Uchl1 mRNA is predominantly localized in the cytoplasm, whereas the natural SINEUP RNA AS-Uchl1 is localized in the nucleus at steady state levels but upon rapamycin treatment it is transported to the cytoplasm to enhance Uchl1 mRNA translation (Carrieri et al, 2012). We hypothesize that the subcellular distribution of SINEUP RNAs has a key role in regulating target mRNA translation. To elucidate the kinetic distribution of EGFP mRNA and SINEUP RNA, we performed RNA FISH (fluorescence in situ hybridization) following co-transfection of EGFP and SINEUP expression vectors (SINEUP-GFP or the deletion mutants) into HEK293T/17 cells. We observed that EGFP mRNAs were predominantly localized in the cytoplasm (Fig 2A, d, i, n, s), whereas the SINEUP RNAs were distributed both in the nucleus and the cytoplasm (Fig 2A, c, h, m, r). In the nucleus, SINEUP RNAs were located throughout the nucleoplasm, but not in the nucleolus. For an unknown reason, SINEUP RNAs formed intensively clustered spots. In the cytoplasm, SINEUP RNAs co-localized with EGFP mRNAs, appearing as numerous small dots distributed throughout the cytoplasm (Fig 2A, u, arrows). Co-localization of EGFP mRNA and SINEUP RNA in the cytoplasm was observed more frequently for SINEUP-GFP (37.75%) and SINEUP-ΔAlu RNAs (30.73%) than for SINEUP RNAs with impaired translational up-regulation activity (i.e., SINEUP-SCR and SINEUP-ΔSB2) ( Fig 2B); SINEUP-∆Alu and SINEUP-GFP showed more frequent overlap peaks (arrows) compared with SINEUP-SCR and SINEUP-∆SB2 ( Fig EV2A). This suggests that BD and ED domains contribute both for the translational up-regulation activity and for the co-localization of EGFP mRNAs and SINEUP RNAs. When the EGFP expression vector was transfected alone, most EGFP mRNA was distributed in the cytoplasm (Fig EV2B), as was observed for co-transfection. In contrast, when the expression vectors for SINEUP RNAs were transfected alone, the SINEUP RNAs were preferably distributed in the nucleus (Fig 2C,e,h,k). To compare the subcellular distribution of SINEUP RNAs between cells co-transfected with EGFP and SINEUP vectors to those transfected with SINEUP alone, the signals were detected from whole cell region and the nucleus region by using icy Spot Detector (Fig EV2C). The percentage of SINEUP-GFP RNA localized in the nucleus was 60.6% when the SINEUP-GFP vector was transfected alone ( Fig 2D); this was significantly reduced to 49.3% and shifted to the cytoplasm when the SINEUP vector was co-transfected with the EGFP vector. A similar finding was observed for SINEUP-ΔAlu, but no significant differences were observed for SINEUP-SCR or SINEUP-ΔSB2 (Fig EV3 A-C). These observations suggest that the SINEUP RNAs with translational up-regulation activity were exported to the cytoplasm in the presence of EGFP mRNA.

Identification and functional analysis of SINEUP RNA binding proteins
We hypothesized that SINEUP RNA binding proteins (RBPs) may play a crucial role in EGFP expression. To identify SINEUP RBPs, we used a modified version of the Chromatin Isolation by RNA Purification (ChIRP) method (Chu et al, 2015) (Fig EV4A, B) followed by mass spectrometry (MS) analysis. By carrying out three or more independent experiments on SINEUP-SCR, SINEUP-∆SB2, SINEUP-ΔAlu and SINEUP-GFP transfection, we detected several SINEUP RBPs. To determine which RBPs are most important for the translational up-regulation activity of SINEUP-GFP, we selected several candidate SINEUP-GFP RBPs with high reliability and specificity (Fig 3) while non-specific bound proteins, which were also detected with beads and LacZ samples, were cut off. We then compared the SINEUP-GFP RBPs with those for the other SINEUP mutants (Fig EV4C-E), and assumed that several nucleocytoplasmic shuttling-related proteins were specifically enriched as SINEUP-GFP RBPs (Fig 3). After excluding ribosomal proteins, we performed siRNA-mediated knockdown of enriched proteins in the scatterplot (Fig 3) to assess their effects on protein up-regulation activity of SINEUP-GFP (Fig 4; Fig EV5A-F). The experiments revealed that knockdown of either PTBP1 (Fig 4A1 and A2) or HNRNPK (Fig 4B1 and B2) significantly decreased the translational up-regulation activity of SINEUP-GFP compared with SINEUP-GFP transfected with scramble siRNA as negative control (Fig 4C1 and C2, SINEUP-GFP ※). Knockdown of EF1A1 decreased the translational-up regulation for EGFP but also affected for β-actin as a non-specific effect (Fig EV5C), suggesting it affected global translational pathways. Interestingly, knockdown of PTBP1 (Fig 4Da-e) and HNRNPK (Fig 4Df-j) significantly reduced the co-localization of EGFP mRNA and SINEUP-GFP RNA in the cytoplasm ( Fig 4E). Furthermore, it also significantly increased SINEUP-GFP RNA nuclear retention: 76.4% for PTBP1 knockdown and 61.4% for HNRNPK knockdown versus 49.3% in control cells ( Fig 4F) suggesting a decrease in cytoplasmic SINEUP RNA; a smaller portion of cytoplasmic SINEUP RNA co-localized with EGFP mRNA. These results suggest that PTBP1 and HNRNPK may participate in the nucleocytoplasmic shuttling of SINEUP RNAs.
To better understand the role of PTBP1 and HNRNPK interactions in SINEUP-GFP RNA nucleocytoplasmic shuttling, we conducted an RNA immunoprecipitation (RIP) assay of RNAprotein interactions with PTBP1 and HNRNPK proteins in the nucleus and cytoplasm. SINEUP-GFP RNAs were pulled down with PTBP1 both in the nucleus ( Fig 5A1) and cytoplasm ( Fig 5A2) and EGFP mRNAs were pulled down with HNRNPK both in the nucleus ( Fig 5B1) and cytoplasm ( Fig  5B2). These observations suggest that (a) PTBP1 protein was able to bind to SINEUP-GFP RNA or the EGFP-SINEUP RNA complex in either the nucleus or the cytoplasm, but not to EGFP mRNA alone; and (b) HNRNPK protein was able to bind to EGFP mRNA or the EGFP-SINEUP RNA complex in either the nucleus or the cytoplasm, but not did bind to SINEUP-GFP RNA alone. Taken together, these findings indicate that PTBP1 and HNRNPK play a role in the formation of the RNA-protein complexes and participate in the kinetic distribution of these RNA-protein complexes.

SINEUP RBPs drive subcellular localization of SINEUP RNAs and participate in translational initiation assembly
We next wondered whether EGFP levels would be further enhanced by overexpression of PTBP1 and HNRNPK proteins. To address this question, we transfected the cells with either PTBP1 or HNRNPK expression vector. The EGFP level was moderately but significantly increased by overexpression of PTBP1 (Fig 6A1 and A3) or HNRNPK (Fig 6B1 and B3) in cells co-transfected with EGFP and SINEUP-GFP vectors, but not in those transfected with EGFP vector alone (Fig 6A2,  B2). This finding suggests that PTBP1 and HNRNPK formed a protein-SINEUP RNA complex and functionally enhanced EGFP levels.
To evaluate the effect of SINEUP RBPs on the subcellular distribution of SINEUP RNA, we overexpressed (or not) PTBP1 ( Fig 7A) or HNRNPK (Fig 7B) in the cells and then co-transfected the cells with EGFP and SINEUP-GFP vectors or with SINEUP-GFP vector alone. Some portion of nuclear SINEUP-GFP RNA was shuttled into the cytoplasm when EGFP and SINEUP-GFP transcripts were co-transfected into cells overexpressing PTBP1 than into cells with normal PTBP1 levels ( Fig 7A2); this difference was not seen when the cells were transfected with SINEUP-GFP vector alone ( Fig 7A3). Induction of PTBP1 did not directly drive SINEUP-GFP RNA from the nucleus to the cytoplasm without the presence of EGFP mRNAs. In contrast to PTBP1, overexpression of HNRNPK had no significant effect on the subcellular distribution of SINEUP-GFP RNAs (Fig 7B2 and B3). Taking these results together, these findings indicate that PTBP1 and HNRNPK participate in nucleocytoplasmic shuttling of RNA-protein complex and play another role after shuttling into the cytoplasm to regulate translation.
To gain further insights into the molecular mechanism of translational enhancement, we analyzed the distributions of the RNAs and RBPs in polysome fractions of cells overexpressing PTBP1 and HNRNPK. The cytoplasmic lysate was separated into 12 fractions by using a 15%-45% sucrose gradient (Fig 8A). EGFP mRNAs were slightly shifted into heavier polysomes when PTBP1 (Fig 8Ba) or HNRNPK (Fig 8Bb) was overexpressed compared with when these SINEUP RBPs were not overexpressed, which is in cells co-transfected with EGFP and SINEUP-GFP vectors (Cont., Fig 8Bc) and in those transfected with EGFP vector alone (Fig 8Bd). SINEUP-GFP RNA co-sedimented with EGFP mRNA in the heavy polysome fractions when EGFP mRNA was present (Fig 8Be, f, g). Although we observed in the FISH analysis (Fig 2C and D) that most SINEUP-GFP RNA was retained in the nucleus when the cells were transfected with SINEUP-GFP alone, more than 85% of the cytoplasmic SINEUP-GFP RNA sedimented in the fractions containing free/40S binding RNAs (47.7%) to monosomes (37.8%) (Fig 8Bh). This implies that the cytoplasmic SINEUP-GFP RNA may participate in an initial phase of translation. We then conducted Western blot analysis of the co-distribution of SINEUP RBPs and the RNAs in the polysome fractions (Fig  8Bi-m). The analysis revealed that HNRNPK co-sedimented with the RNAs and PTBP1 in the light polysome fractions when HNRNPK was overexpressed (Fig 8Bj). PTBP1 also co-sedimented with the RNAs in the light polysome fractions when PTBP1 was overexpressed (Fig 8Bi). Additionally, PTBP1 co-sedimented with SINEUP-GFP RNA from the free/40S fractions to the monosome fractions even when SINEUP-GFP vectors were transfected alone (Fig 8Bm). This indicates that SINEUP RNAs associate with PTBP1 and may recruit ribosome subunits to contribute to the formation of translational initiation complexes, including elongation factor EF1A, to participate in the initial phases of translation.
To investigate direct protein-protein interactions, we performed BS3 chemical cross-linked immunoprecipitation. The results revealed that PTBP1 and HNRNPK directly interacted with each other both in the nucleus and the cytoplasm (Fig EV6A, B), and PTBP1 and HNRNPK also had direct interactions with 40S and 60S ribosome subunits in the cytoplasm, suggesting that these SINEUP RBPs form a complex with SINEUP RNAs and EGFP mRNAs, and participate together in nucleocytoplasmic shuttling and translational regulation.

Discussion
In this study, we demonstrated that a synthetic SINEUP RNA plays a critical role in the enhancement of the translation of its target mRNA by 1) co-localizing with the target mRNA in the cytoplasm, 2) interacting with SINEUP RBPs to influence the SINEUP RNA's distribution and 3) increasing target mRNA shifting to polysome by participating in translational initiation assembly.
By conducting RNA FISH, we revealed that the co-localization of EGFP mRNA and SINEUP-GFP RNA in the cytoplasm was required for positive translational regulation. We previously reported that the AS-Uchl1 is enriched in the nucleus and exported to the cytoplasm under rapamycin-induced stress, which inhibits the mTOR (mammalian target of rapamycin) pathway, resulting in up-regulation of UCHL1 translation (Carrieri et al, 2012); however, in our subsequent study, the distribution of synthetic SINEUP RNAs in the cytoplasm was not increased by inhibition of the mTOR pathway (Zucchelli et al, 2015). Here, we demonstrated that the synthetic SINEUP-GFP RNA localized both in the nucleus and the cytoplasm when EGFP mRNA and SINEUP RNA co-existed, but was retained in the nucleus in the absence of EGFP mRNA. This suggests that synthetic SINEUP RNAs may have specific export systems that rely on specific unknown motifs for SINEUP-binding factors, and which differ from the mTOR-dependent export system of AS-Uchl1. From loss-and gain-of-function studies of SINEUP RBPs, we found that PTBP1 and HNRNPK proteins have key roles in SINEUP-GFP RNA nucleocytoplasmic shuttling and in up-regulating translation. PTBP1 (also known as HNRNPI) is a multifunctional RNA binding protein that participates in alternative splicing, mRNA stabilization, and nucleocytoplasmic shuttling by binding to polypyrimidine-rich tract in pre-mRNAs (Singh et al, 1995;Sawicka et al, 2008;Romanelli et al, 2013). PTBP1 is known as a binding factor for TOP mRNA, which contains 5' terminal oligopyrimidine tract (5'TOP) mostly found in mRNAs encoding ribosomal protein and elongation factors (Avni et al, 1997;Meyuhas, 2000), to regulate translation of the target TOP mRNA (Gismondi et al, 2014) as a cis-acting regulator . LARP1, which is known as 5'TOP mRNA binding proteins, stabilize the target 5'TOP mRNAs by forming complex with 40S ribosome subunit (Gentilella et al, 2017), In this study, SINEUP-GFP showed direct interaction with 40S, implying that SINEUP RNA with multimerization including PTBP1, 40S and the target mRNA may contribute the target mRNA stabilization. There are several reports that PTBP is recruited to cap-independent translation to stimulate translational initiation with re-modelling the target transcripts followed by supplying the small subunit (40S) to binding sites (Caceres et al, 2016;Stoneley & Willis, 2004). In one example of translational initiation mechanism, the cricket paralysis virus (CrPV) recruits 40S ribosome subunit on its RNA with high affinity, and requires eukaryotic elongation factor (eEF)1A and eEF2 to recruit tRNA for assembly of 80S tertiary complexes (Johnson et al, 2017). One of the key findings in this study, several components of ribosomal complexes such as eEF1A, eEF2 and ribosome proteins were also detected as SINEUP RBPs (Fig 3), and EF1A and PTBP1 were co-sedimented with the RNAs in various fractions ranging from free/40S-binding RNA to light polysome fractions. This indicates that SINEUP-GFP RNA may become a scaffold to bind PTBP1, and to recruit translational initiation factors; this likely supplies the 40S subunit to mRNA binding sites, thereby helping initiation assemblies ( Fig 8C). Although we need to further characterize the protein-protein interactions, our data suggest that complexes of SINEUP RBPs plus SINEUP RNAs affect translational regulation in the initiation phases.
HNRNPK has three KH (K homology) domains, which bind RNAs, and unique nuclear localization signals which have bi-directional transport, that enable to export the nuclear envelope with target mRNAs (Michael et al, 1997). HNRNPK regulates the target mRNA's translation positively or negatively, depending on the target mRNA. As an example of positive regulation, HNRNPK bound to VEGF mRNA and stimulated the ribosome to bind the mRNA resulting in a shift to heavier polysomes (Feliers et al, 2007). In contrast, in a case of negative regulation, HNRNPK blocked monosome assembly by binding to the 3ʹ UTR of c-Src mRNA thereby repressing the translation (Naarmann et al, 2008). In our study, HNRNPK shifted to heavier polysomes with EGFP mRNA when HNRNPK was overexpressed and up-regulated EGFP mRNA translation, suggesting positive regulation; HNRNPK contributed to ribosome assembly only when EGFP mRNA and SINEUP-GFP RNA co-existed.
When PTBP1 or HNRNPK were overexpressed, EGFP enhancement was observed only when SINEUP-GFP RNA and EGFP mRNA coexisted, i.e., not when the cells were transfected with EGFP construct alone. Similarly, when PTBP1 was overexpressed, SINEUP-GFP RNA distribution was affected only when SINEUP-GFP RNA and EGFP mRNAs co-existed. These findings imply that the SINEUP RBPs, SINEUP-GFP RNA, and EGFP mRNAs together form RNA-protein complexes, and that these multiple components are essential for a functional complex. Several studies report protein-protein direct interaction between PTBP1 and HNRNPK, suggesting that they function cooperatively in biological processes (Kim et al, 2000). One lncRNA, TUNA, which is known to maintain pluripotency in neuronal cells, to form complexes including PTBP1 and HNRNPK, associates with Nanog, Sox2, and Fgf4 to activate these pluripotency genes (Lin et al, 2014). Lncenc1, a highly abundant lncRNA in naive embryonic stem cells, recruits PTBP1 and HNRNPK and to bind to glycolysis genes, thereby regulating cell pluripotency and glycogenesis (Sun et al, 2018). Taken together, our results and these reports indicate that formation of RNAprotein complexes containing SINEUP-GFP RNAs and RBPs such as PTBP1 and HNRNPK affects not only nucleocytoplasmic shuttling of the SINEUP-GFP RNAs, but also participates in translational initiation assembly to recruit initiation factors for up-regulating translation of the target mRNA ( Fig 8C).
Interestingly, both HNRNPK and PTBP1 are classed as heterogeneous nuclear ribonucleoproteins (RNPs), which mainly participate in alternative mRNA splicing, conformation of RNP assembly to compact transcripts in the nucleus, and nucleocytoplasmic shuttling (Dreyfuss et al, 1988). RNA-RNA intermolecular interactions in RNP granules, which are non-membrane-bound organelles including specific RBPs, contribute to various cellular functions (Van Treeck & Parker, 2018). Here, we observed that some of the SINEUP-GFP RNA was retained in the nucleus and co-localized with the RBPs, suggesting that the SINEUP RNA and RBPs may form RNA-protein granules that accrue in the nucleus as part of a yet-unknown mechanism for RNA modification and editing. Some reports suggest that nuclear history determines a transcript's fate in the cytoplasm (Matsumoto et al, 1998), and the exon junction complex, which includes splicing factors such as RNPs, affects mRNA destination (Giorgi & Moore, 2007).
Although the binding domain and structural conformation of binding factors in SINEUPprotein interactions require further study, our results suggest that intermolecular interactions between SINEUP RNAs and RBPs contribute to the translational up-regulation of the target mRNA. This improved understanding of the mechanisms of efficient protein translational regulation by functional lncRNAs, and will help to facilitate broad applications of RNA regulation such as nucleic-acid-based therapies.

Plasmid transfection and conditions
The pEGFP-C2 and SINEUP vectors were co-transfected into HEK293T/17 cells by using Lipofectamine 2000 (Invitrogen) with OptiMEM (1×) Reduced Serum Medium (Gibco). After testing EGFP mRNA translation at various time points after transfection, we confirmed that up-regulation of translation of EGFP mRNA occurred from 24 h to 48 h post-transfection.

Measuring protein up-regulation by Western blot assay.
Cells were plated in six-well plates, transfected with plasmid(s), lysed in Cell Lysis buffer (Cell Signaling), and incubated at 4°C for 1 h. The cell lysates were applied to 10% precast polyacrylamide gels (Bio-Rad) for SDS-PAGE and transferred to nitrocellulose membranes (Amersham). The membranes were incubated for 1 h at room temperature with the primary antibody, anti-GFP rabbit polyclonal antibody (1:1000 dilution; A6455, Thermo Fisher Scientific), and then for 45 min at room temperature with the secondary antibody, anti-rabbit IgG conjugated with HRP (P0448, Dako), and EGFP was detected by ECL detection reagent (Amersham). As a control, anti-β actin mouse monoclonal antibody (1:1000 dilution; A5441, Sigma Aldrich) was used as the primary antibody, and anti-mouse IgG-conjugated HRP (1:1000 dilution; P0447, Dako) was used as the secondary antibody.

RNA extraction and reverse transcription real-time quantitative PCR (RT-qPCR)
RNAs were extracted with an RNeasy mini kit (QIAGEN) following the manufacturer's instructions. The TURBO DNA-free Kit (Invitrogen) was used for DNase I treatment to remove plasmid DNAs. For RT-qPCR, cDNA was synthesized by using the PrimeScript 1st strand cDNA synthesis kit (TAKARA), and PCR was performed with SYBR Premix Ex Taq II (TAKARA) and the 7900HT Fast Real-Time PCR System (Applied Biosystems). The thermocycling protocol was 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s.

RNA FISH
FISH probes for target transcripts were designed by using Stellaris RNA FISH designer (BIOSEARCH Technologies; https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer), and fluorescently labelled with Quasar 570 (for SINEUP RNAs) or Quasar 670 (for EGFP mRNA). Cells were fixed with 4% paraformaldehyde (WAKO) and permeabilized with 0.5% Triton X-100 (Sigma) at room temperature for 5 min. Hybridization was performed overnight at 37°C. Nuclei were visualized by incubation with Hoechst 33342 (H3570, Thermo Fisher Scientific). After sequential washing steps, cell images were detected by using a SP8-HyVolution confocal laser scanning microscope (Leica Microsystems) with a 63x/1.4 oil objective lens; the images were processed using HyD detectors with Huygens Essential software (Scientific Volume Imaging  Lagache et al, 2015).

Detection of SINEUP RBPs
The protocol for detecting SINEUP RBPs was based on the protocol for the Magna ChIRP RNA Interactome kit (Merck Millipore) with the modification of cross-linking with 300 mJ/cm of 264 nm UV light (CL-1000 Ultraviolet Cross Link, UVP). The cell pellet (2 × 10 7 cells) was suspended wit 2 mL of Lysis buffer (Cell Signaling) supplemented with Protease Inhibitor Cocktail Set III (Merck Millipore), mixed by rotation at 4°C for 30 min, and then sonicated for 8 cycles (ON for 30 s, OFF for 30 s) using a Picoruptor Sonicator (Diagenode). Each tube of lysate was incubated overnight at 37°C in hybridization buffer (750 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA) with the addition of 15% v/v formamide (Sigma), Phenylmethanesulfonyl Fluoride (PMSF) (Cell Signaling), Protease Inhibitor Cocktail Set III (Merck Millipore) and SUPERase• In RNase Inhibitor (Thermo Fisher Scientific) just before use, and 100 pmol of probe. Each lysate was then incubated at 37°C for 30 min with washed MagCapture Tamavidin 2-REV magnetic beads (WAKO). After sequential washes, the bead samples were separated into two halves for protein and RNA extraction. Proteins were extracted as reported previously (Chu et. al, 2015); the eluent was incubated in DNase/RNase solution (100 μg/mL RNase A, 0.1 U/μL RNase H, and 100 U/mL DNase I) at 37°C for 1 h followed by acetone precipitation. The protein samples were digested with 10 ng/μL Sequencing Grade Modified Trypsin (V5111, Promega) overnight, and the resultant peptides were subjected to Liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the Support Unit for Bio-Material Analysis, Research Resource Center, Brain Science Institute in Wako, Japan. Proteome Discover (version 1.4) software with the MASCOT search engine (version 2.6.0) was used in the Swiss-Prot database. For RNA extraction, the beads were incubated with proteinase K at 55°C overnight, and then extracted with Trizol (Thermo Fisher Scientific) and chloroform (WAKO). The eluent was treated with DNase I (Ambion), and the RNA expression level was quantified by RT-qPCR.

RIP
was performed with the Abcam protocol (https://www.abcam.com/epigenetics/rna-immunoprecipitation-rip-protocol) with some modifications. The cells were plated into 10-cm plates, and followed by plasmid transfection described above, and then next day, nuclei and cytoplasmic fractions were isolated. Nuclear pellets were sheared by sonication with 5 cycles (ON for 30 s, OFF for 30 s) using a Bioruptor Pico device. To immunoprecipitate RNA with the antibodies for target proteins, each lysate was incubated at 4°C overnight. Then Protein A/G magnetic beads (Invitrogen) were added to bind the target antibodies, and sequential washing was conducted to remove unbound antibodies. The anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade (ab39975, Abcam) and anti-PTBP1 mouse monoclonal antibody (32-4800, Thermo Fisher Scientific) were used to immunoprecipitate HNRNPK and PTBP1, respectively, in each nuclear or cytoplasmic fraction. To purify the RNA, the lysates were incubated with protease K at 55°C overnight followed by Trizol (Thermo Fisher Scientific) and chloroform (WAKO) extraction. RNA levels were quantified by RT-qPCR.

Immunofluorescence microscopy
The cells were prepared as described above for RNA FISH. After the cells were permeabilized, the primary antibodies, anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade (ab39975, Abcam) and anti-PTBP1 mouse monoclonal antibody (32-4800, Thermo Fisher Scientific), were added and hybridized overnight. Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibodies (A-21236, Thermo Fisher Scientific) were used to visualize the results.

Polysome fractionation
Polysome fractionation was performed as reported previously (Faye et al, 2014). Briefly, 2.5 × 10 6 cells were plated into 10-cm plates, 18h later, followed by hnRNPK and PTBP1 clone vector transfection, then additional 6 h later, EGFP and SINEUP vector transfection were performed as described above. The transfected cells were maintained in growth media without antibiotics for 48 h after the clone vector transfection, then incubated with 0.1 mg/mL cycloheximide for 5 min at 37°C followed by washing with ice-cold PBS containing 0.1 mg/mL cycloheximide. The harvested cells were centrifuged at 300 × g for 10 min at 4°C. The cell pellets were suspended with 200 μL of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 30 mM MgCl 2 , 0.1 mg/mL cycloheximide, 0.1% NP-40, with fresh RNase inhibitor and Proteinase inhibitor cocktail added just before use). The cell lysate was incubated for 10 min on ice followed by centrifugation at 2,000× g at 4°C for 5 min to separate the nuclei. The cytoplasmic fraction was subjected to further centrifugation at 17,000× g at 4°C for 5 min to remove cell debris. The cytoplasmic lysate was layered onto a 15%-45% sucrose gradient and centrifuged in an SW41Ti Beckman rotor at 190,000× g at 4°C for 3.5 h. The sucrose gradient was separated into 12 fractions calculated by Triax flow cell (Biocomp). Half of each fraction was treated with Proteinase K at 55°C overnight then followed by Trizol and chloroform extractions as described above. The eluent was treated with DNase I (Ambion), and the RNA expression level was quantified by RT-qPCR. Using the other half of each fraction, proteins were isolated using the Thermo Fisher Scientific acetone precipitation protocol: 4 volumes of cold acetone were added to each sample, and then the samples were incubated at -20°C for 1 h or overnight, and centrifuged at 13,000 × g for 10 min at 4°C. Each pellet was suspended in PBS and subjected to Western blot analysis.

Protein-protein direct interaction with chemical cross-linking
BS3 (bis [sulfosuccinimidyl] suberate) cross-linking was performed based on a published protocol (de la Parra et al, 2018). The cells were incubated with 0.6 mM BS3 (Thermo Fisher Scientific) for 30 min; the reaction was quenched by incubation with 1M Tris-HCl (pH 7.5) for 15 min at room temperature. The cell lysate was incubated with target antibodies as described for RIP. After sequential washes, the beads were incubated with 2× Laemmli sample buffer (Bio-Rad) at 95°C for 20 min to dissociate proteins, followed by Western blot assay.

Statistical analysis
Statistical differences were measured using Student's t-test with paired, two-sided. Bar graphs were described as means ± standard deviation (SD) from at least 3 independent experiments. Statistically significances were represented with *p < 0.05, **p < 0.01. To test normality of all data sets, Kolmogorov-Smirnov test was used.  C Quantification of the up-regulation of EGFP by co-transfection with EGFP and SINEUP vectors. **p < 0.01, ns: not significant, by Student's t-test. Data are means ± SD from at least 3 independent experiments. D Quantification of the EGFP mRNA levels following co-transfection with EGFP and SINEUP expression vectors. Data are means ± SD from at least 3 independent experiments.

Figure 2 Co-localization of SINEUP-GFP RNAs with EGFP mRNAs in the cytoplasm
A Subcellular localization of SINEUP RNAs and EGFP mRNAs. Bars indicate 5 μm.
B Comparison of percentage co-localization of EGFP mRNAs and SINEUP RNAs in the cytoplasm. Data are means ± SD of at least 10 independent cell images. **p < 0.01, ns: not significant by Student's t-test.
C Subcellular distribution of SINEUP RNAs following transfection with SINEUP expression vectors alone. Bars indicate 5 μm. D Quantitative comparison of the distribution of SINEUP-GFP RNA in the presence and absence of EGFP mRNA. The ratios of detected spots in the nucleus and cytoplasm were compared between co-transfection of SINEUP-GFP and EGFP vectors, and transfection of SINEUP-GFP vector alone. Data are means ± SD of at least 10 independent cell images. **p < 0.01, ns: not significant by Student's t-test.

Figure 3 SINEUP-GFP RNA binding proteins
Detected RNA binding proteins (RBPs) were plotted according to relative reliability ( ) on the x axis, and relative specificity ( ) on the y axis. Proteins that were detected by beads and LacZ probes were omitted. For both PTBP1 and HNRNPK, the ratio of SINEUP-GFP RNA levels in the nucleus and the cytoplasm were compared between the knockdowns and the negative control. **p < 0.01 by Student's t-test. Data are means ± SD of at least 10 independent cell images.

Figure 5 RNA immunoprecipitation
A1, A2 RIP with PTBP1 antibody in the nucleus (A1) and cytoplasm (A2). Isotype IgG was utilized as the negative antibody control. As negative target RNAs, transcripts from the housekeeping genes, MALAT1 and GAPDH were utilized as controls in the nucleus and cytoplasm, respectively. *p < 0.05, **p < 0.01 by Student's t-test. Data are means ± SD of at least 3 independent experiments. B1, B2 RNA immunoprecipitation (RIP) with HNRNPK antibody or isotype immunoglobulin G (Isotype IgG; negative control) in the nucleus (B1) and cytoplasm (B2). The cell lysate co-transfected pEGFP-C2 and SINEUP-GFP vectors or that transfected ether vector alone were tested.  A2, A3, B2, B3 Quantitative comparison of SINEUP-GFP RNA distribution between cells overexpressing PTBP1 (A2, A3) or HNRNPK (B2, B3) and non-overexpressing these proteins (Cont.). Results for cells co-transfected with EGFP and SINEUP-GFP vectors (A2 and B2) and those transfected with SINEUP-GFP vector alone (A3, B3) are shown. SINEUP RNA signals were detected using Icy Spot Detector. The ratio of spots in the nucleus and the cytoplasm were compared between overexpression and non-overexpression of SINEUP RBPs. *p < 0.05 by Student's t-test.
Data are means ± SD of at least 10 independent cell images.

Figure 8 RNA distribution in polysome fractions obtained from cells overexpressing SINEUP RBPs
A Representative polysome gradient profile with optical density (290 nm). Fractions ① and ⑫ correspond to the 15% and 45% sucrose fractions, respectively.
B Polysome profiling of EGFP mRNA (green) and SINEUP-GFP RNA (blue). RNA distribution was quantified by RT-qPCR. Each fraction of EGFP mRNA in cells co-transfected with EGFP and SINEUP-GFP vectors (a,b,c) was compared with the corresponding fraction in cells transfected with EGFP vector alone (d). *p < 0.05, **p < 0.01 by Student's t-test. Data are means ± SD from 3 independent experiments. The protein distribution in polysome fractions with non-/overexpressed PTBP1 or HNRNPK were tested (i-m). Equal volumes of solution were pooled as Free/40S (Free or 40S binding RNA fraction from ①-③ at A), Mono (monosome fraction from ④-⑥), Light (light polysome fraction from ⑦-⑨), and Heavy (Heavy polysome fraction from ⑩-⑫) and were applied to 10% SDS gels to detect each target protein. Western blot images representative of least 3 independent experiments are shown.
C Model of SINEUP RNA and SINEUP RBP interactions. SINEUP RBPs (PTBP1 and HNRNPK) participate in SINEUP RNA localization both in the nucleus and the cytoplasm. In the nucleus, some portion of the RNAs form RNA-protein granules (dotted circle) where probably immature transcripts accumulate to cluster non-proper conformation, and the other portion of mature transcripts including EGFP mRNA and SINEUP RNA form complex with SINEUP RPBs, which may be shuttled into the cytoplasm. In the cytoplasm, SINEUP RNAs co-operate with the SINEUP RBPs likely re-modelling SINEUP RNA structure, and recruit ribosomal subunits to efficiently supply them to EGFP mRNA, resulting into positive translational enhancement of EGFP mRNA. EGFP mRNA can be exported into the cytoplasm by itself, but its translation is initiated more efficiently when SINEUP-GFP RNA is present in the cytoplasm. B Subcellular localization of EGFP mRNAs following transfection with EGFP expression vectors alone. Bars indicate 5 μm.

Expanded View Figure legends
C Spot detected images in the cells with co-transfection and with SINEUP-GFP alone transfection. Using icy Spot Detector, signals were detected from the whole cell region and from the nucleus region. Bars indicate 5 μm.

Fig EV3. Comparison of subcellular distribution of SINEUP mutants in cells transfected with SINEUP RNA vector alone and cells co-transfected with EGFP and SINEUP RNA vectors
A, B, C The ratio of cytoplasmic distributions of the SINEUP mutants SINEUP-SCR (A), SINEUP-∆SB2 (B) and SINEUP-∆Alu (C) were compared with and without transfection of EGFP vector. *p < 0.05, ns: not significant by Student's t-test. Data are means ± SD of at least 10 independent cell images.
B Percentage retrieval of RNA by modified ChIRP with SINEUP RNA probe. Biotinylated SINEUP-GFP probe specifically enriched SINEUP-GFP RNA compared with the Magna ChIRP negative control probe (LacZ) and magnetic beads alone (probe (-)). As a negative target RNA, GAPDH mRNA was tested to assess non-specific interaction.

Fig EV5. Knockdown of SINEUP RBPs.
A-F Knockdown of SINEUP RBPs. Numbers at the top row under the image show knockdown efficiency compared with cells transfected with the SINEUP-GFP vector and negative control siRNA (SINEUP-GFP※ in Fig 4C1 and C2). *p < 0.05, **p < 0.01, ns: not significant by Student's t-test. Data are means ± SD of at least 3 independent experiments.

Fig EV6. Protein-protein direct interaction by chemical crosslinking with bis (sulfosuccinimidyl) suberate (BS3).
A, B Representative Western blot images of immunoprecipitation (I.P.) with PTBP1 antibody (A) or HNRNPK antibody (B). Isotype IgG was utilized as the negative antibody control.