Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP‐based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell‐free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.

The advancement of genomics technologies revealed that an emerging class of long noncoding RNAs (lncRNAs) [1], which constitute the majority of types of transcripts and do not encode proteins [2,3], plays key regulatory roles in the physiology of normal cells, the development of diseases including cancer [4] and neurodegenerative diseases [5]. The discovery of increasing numbers of functional lncRNAs has prompted novel therapeutic applications, including the treatment of human genetic diseases. A functional lncRNA transcribed from the antisense strand of Uchl1 mRNA contains an inverted SINEB2 element and upregulates the translation of its sense strand Uchl1/Park5 mRNA in mouse dopaminergic neuronal cells [6]. This lncRNA was the first in a large class of antisense lncRNAs, named 'SINEUPs', because they contain an embedded inverted SINEB2 element that acts as an effector domain (ED) to mediate UPregulation of the translation of the target mRNA; the target specificity is determined by the antisense RNA region, the binding domain (BD) [7].
SINEUP-based upregulation systems occur in various cell types [7] and vertebrates including humans, mice, and fish [8][9][10][11][12]. We have focused on broadening the scope of SINEUP-based upregulation systems so that they can be applied to direct nucleic acid-based therapeutics [5,13,14]. We consider that SINEUPs might be therapeutically useful for various disorders caused by insufficient protein production [10] or haploinsufficiency. At least 300 genes are linked to haploinsufficiencies [15], for which more than 3000 genes are predicted as possible therapeutic candidates [16]. In one SINEUP proof-of-concept study, the target is the medaka cox7B gene; mutations in the human homolog subunit 7B of cytochrome c oxidase (COX7B), the product of which is a component of the mitochondrial respiratory chain, are responsible for the disease microphthalmia with linear skin lesions. To rescue a medaka model of microphthalmia with linear skin lesions, we introduced synthetic in vitro transcribed (IVT) SINEUP-cox7B, designed against endogenous cox7B mRNA. IVT SINEUP-cox7B enhanced COX7B protein production and consequently rescued eye and brain size in cox7B morphants [12]. Whether direct administration of SINEUP RNA effectively enhances protein production in cell types or species other than medaka is unknown. Here, we successfully developed synthetic, chemically modified IVT (mIVT) SINEUPs that upregulated the translation of target enhanced green fluorescent protein (EGFP) mRNA and endogenous target sex-determining region Y (SRY)-box 9 (Sox9) mRNA in cultured cells. In addition, mIVT SINEUP RNA successfully upregulated EGFP production in a HeLa extract cell-free translation system, which contains the SINEUP-specific RNA-binding proteins (RBPs) HNRNPK and PTBP1 [17].
The current study improves our understanding of the system through which mIVT SINEUP RNAs regulate both exogenous and endogenous targets in specific cell types and in a cell-free system. Therefore, our current findings support nucleic acid-based therapeutics as additional tools for gene therapy of human disorders due to insufficient protein production.

Plasmids and constructs
The pEGFP-C2 plasmid was purchased from Clontech Laboratories (Takara Bio USA, Mountain View, CA, USA). The pCS2+_SINEUP-GFP plasmid was generated in a previous study [17]. The BD of SINEUP targeting GFP, D5 0 -32 nt, has a deletion of 28 bases from the 5 0 end of the original 60 nt SINEUP-GFP and corresponds to the mRNA positions À28 to +4 (see Fig. 1B in [18]). The pcDNA3.1_EGFP plasmid was constructed by cloning a fragment encoding full-length EGFP (À40 bp to the stop codon) from the plasmid pEGFP-C2 into pcDNA3.1(-) (Thermo Fisher Scientific, Waltham, MA, USA). The SINEUP targeting mouse Sox9 (named miniSINEUP-Sox9) contained a BD that overlapped mouse Sox9 mRNA (in antisense orientation) and the control without BD (named miniSINEUP-Random; Rd) contained a random sequence instead of the Sox9-BD, those have an ED containing an inverted SINEB2 sequence from mouse AS-Uchl1 RNA, were cloned into the pCS2+ vector (Fig. S1).

Plasmid and RNA transfection
HEK293T/17 cells were plated into 12-well plates (1 9 10 5 cells per well), followed 24 h later by transfection of plasmid or RNA (IVT, or mIVT). To detect EGFP, 1380 ng SINEUP-GFP plasmid or 720 ng (m) IVT SINEUP-GFP RNA was cotransfected with 300 ng pEGFP-C2 in each well by using Thermo Fisher Scientific. In vitro translation was performed according to the manufacturer's protocol. Briefly, for each reaction, 400 ng of SINEUP plasmid or 200 ng of (m)IVT SINEUP RNA was mixed with 120 ng pcDNA3.1_EGFP. The mixture was incubated for 90 min at 30°C. Protein expression was measured by western blotting.

Western blotting
Transfected cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and incubated at 4°C for 1 h. Cell lysates were loaded on a 10% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA), separated by SDS/PAGE, and transferred to a nitrocellulose membrane (Amersham, Chicago, IL, USA). All primary and secondary antibodies were used at 1 : 1000 dilution. To detect EGFP, anti-GFP mouse monoclonal antibody (clone JL-8; catalog no. 632380; Clontech, Chicago, IL, USA) in the RRL cellfree system and anti-GFP rabbit polyclonal antibody (catalog no. A-6455; Thermo Fisher Scientific) in a lysate of HeLa cells and in culture cells were used. To detect RBPs, 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 used. To detect endogenous SOX9, anti-Sox9 rabbit monoclonal antibody (clone EPR14335; catalog no. ab185230; Abcam) was used. The membranes were incubated with primary antibodies at 4°C overnight, followed by incubation for 45 min at room temperature with secondary antimouse IgG, or anti-rabbit IgG conjugated with HRP (Dako, Tokyo, Japan). Bands were visualized by using ECL Detection Reagent (Amersham). As a control, primary mouse anti-b actin monoclonal antibody (Sigma-Aldrich) and secondary HRP-conjugated antimouse IgG (Dako) were used. Bands were detected by using the quantification analysis module and chemiluminescence application protocol of the Fusion Solo S System (Vilber-Lourmat, Osaka, Japan).

RNA extraction and quantification
Total RNA was extracted by using RNeasy Mini kit (Qiagen), followed by DNase I treatment (TURBO DNA-free kit; Invitrogen). cDNA was synthesized by using PrimeScript 1st Strand cDNA Synthesis kit (Takara), and quantitative real-time PCR analysis was performed by using SYBR Premix Ex Taq II (Takara) in a model 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
Thermocycling conditions consisted of an initial 30 s at 95°C, 40 cycles comprising 95°C for 5 s, and 60°C for 30 s, followed by melting curve analysis.

RNA fluorescence in situ hybridization
RNA fluorescence in situ hybridization (FISH) was performed as previously described [17]. Briefly, cells were fixed in 4% paraformaldehyde (Wako) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 5 min. RNA was hybridized with fluorescently labeled (Quasar 570 for SINEUP RNAs and Quasar 670 for EGFP mRNA) RNA FISH probes designed by using Stellaris RNA FISH Designer (Biosearch Technologies, Novato, CA, USA; Tables S1 and S2) and incubated overnight at 37°C. Cells were washed and imaged by using a model SP8 (Leica, Wetzlar, Germany) confocal microscope.

Results
IVT SINEUP-GFP RNA failed to increase EGFP synthesis Previously, we found that colocalization of target mRNA and SINEUP RNA in the cytoplasm is required for upregulation of target mRNA translation [17]. We therefore hypothesized that direct transfection of IVT SINEUP RNA into the cytoplasm would enhance protein production more than transfection of SINEUP plasmid, which requires the export of RNA transcribed in the nuclei. To test the efficiency of translational upregulation by using IVT SINEUPs, we transfected IVT RNA into HEK293T/17 cells, with EGFP plasmid as a target sense transcript. IVT SINEUP-GFP RNA contains a BD designed to target EGFP mRNA (Fig. 1A), whereas IVT SINEUP-SCR RNA was designed as a negative control and contains a scrambled, nonfunctional EGFP BD. Transfection of IVT SINEUP-GFP did not stimulate the translation of EGFP (Fig. 1B), although, consistent with previous studies, EGFP mRNA and SINEUP levels did not differ among the cells (Fig. 1C).
In RNA FISH, most of the IVT SINEUP RNAs aggregated into intense spots within cells, or it was difficult to detect IVT SINEUP RNAs at all (Fig. 1D,e, j). These findings suggested that IVT SINEUP RNAs were partially degraded immediately after transfection and were detected as RNA fragments or were not present in sufficient quantities to adequately colocalize with EGFP mRNA. Consequently, EGFP translation was not upregulated.

IVT SINEUP RNAs in cells need to be stabilized through nucleotide modification
Because RNA FISH experiments showed that IVT SINEUPs were likely aggregated after transfection into cells, we hypothesized that stabilization of IVT SINEUP-GFP was needed to enhance EGFP mRNA translation. RNA transcripts can be stabilized by modified nucleotides, such as m 5 C [20,21], Ψ [22,23], or N 1 mΨ [24][25][26], and these modifications contribute to efficient translation. We therefore synthesized mIVT SINEUP RNAs in which all C or U were replaced with the modified forms and transfected them, together with EGFP plasmid, directly into HEK293T/17 cells ( Fig. 2A). All mIVT SINEUP-GFPs increased EGFP production compared with the control (EGFP alone and mIVT SINEUP-SCRs; Fig. 2B), without affecting EGFP mRNA levels (Figs. 2C, Fig. S2A).
We previously found that SINEUP-GFP RNAs localized both in the nucleus and in the cytoplasm when pEGFP-C2 and SINEUP-GFP plasmids were cotransfected [17]. We now found that (m)IVT SINEUP-GFP localized in the cytoplasm regardless of EGFP plasmid transfection (Fig. 2D, Fig. S3A). Upregulation of EGFP mRNA translation did not significantly affect EGFP mRNA levels (Fig. 2C, Fig. S2A). Notably, the RNA levels of mIVT SINEUP RNAs were more than 1.5-fold greater than those of IVT SINEUP RNAs (Fig. S2B), implying that these modified nucleotides contributed not only to EGFP upregulation but also to the stabilization of SINEUPs in cells.

mIVT SINEUPs contribute to EGFP upregulation with SINEUP-binding proteins
We used cell-free translation systems to observe SINEUP upregulation activity separately from RNA stabilization. None of the SINEUP RNAs tested upregulated EGFP in RRL (Fig. 3A), whereas mIVT SINEUPs with Ψ and N 1 mΨ-upregulated EGFP in HeLa cell lysate (Fig. 3B). This result suggests that modified nucleotides contribute to the upregulation of EGFP in cell-free system, implying that the expression of cellular components, including RBPs, is important for SINEUP activity [17]. Indeed, the SINEUP RBPs PTBP1 and HNRNPK were expressed at lower levels, approximately 0.2-and 0.5-fold, respectively, in RRL compared with HEK293T/17 cell lysate, which did not show upregulation of EGFP (Fig. 3C-E). These findings suggest that PTBP1 and HNRNPK may have a role in the SINEUP-based upregulation of translation.
mIVT SINEUPs enhance endogenous SOX9 target protein production To test whether SINEUPs can increase production of endogenous target proteins, we focused on sex-determining region Y (SRY)-box 9 (SOX9) protein; this transcription factor regulates cell differentiation, development, and gene expression in several tissues and organs in vertebrates [27]. In addition, SOX9-positive cells in adult liver can regenerate as hepatocytes after injury [28,29]. Because SINEUPs ultimately are to be used in therapeutic applications, small but still functional SINEUPs are desirable [7]. We therefore designed the miniSINEUP-Sox9 plasmid, in which the BD (À31/+4) overlapped the mouse Sox9 mRNA and miniSINEUP-Rd plasmid, in which contains a random sequence, nonbinding domain instead of the Sox9-BD, those contained the inverted SINEB2 ED from AS-Uchl1 RNA (Fig. 4A, Fig. S1). We then transfected these plasmids into HepG2 and Hepa 1-6 cells to examine enhancement of SOX9 protein production. Cells transfected with the miniSINEUP-Sox9 plasmid showed an approximately 1.5-fold upregulation of SOX9 protein compared with the control (no SINE-UPs) at both 24 and 48 h after transfection in HepG2 cells and at 24 h after transfection in Hepa 1-6 cells (Fig. 4B). In addition, the SINEUP RBPs PTBP1 and HNRNPK were expressed in both HepG2 and Hepa 1-6 cells, at no lower than 0.6 times the level in HEK293T/17 cells (Fig. S4A-C). Consistent with  EGFP study regarding EGFP mRNA, endogenous Sox9 mRNA levels did not change in SINEUP-transfected cells (Fig. 4C). Together, these findings show that miniSINEUP-Sox9 can effectively increase endogenous SOX9 protein levels.
We next tested the efficiency of translational upregulation when mIVT miniSINEUP-Sox9 containing m 5 C, Ψ, or N 1 mΨ was used (Fig. 5A) in HepG2 cells. Similar to the miniSINEUP-Sox9 plasmid, mIVT miniSI-NEUP-Sox9 RNAs with Ψ and N 1 mΨ showed approximately 1.5-fold upregulation of SOX9 protein compared with the control (no mIVT SINEUPs; Fig. 5B). Consistent with the results of plasmid transfection, the endogenous Sox9 mRNA level was not affected by mIVT miniSINEUP RNAs (Fig. 5C). This result implies that nucleotide modifications might contribute to the stabilization of miniSINEUP-Sox9 RNA to enhance the SOX9 protein level in HepG2 cells.

Discussion
Synthetic IVT RNAs have diverse potential applications as a new class of druggable tools based on nucleic acid therapeutics. Here, we demonstrated an efficient approach to enhance target protein production through direct transfection of synthetic chemically modified SINEUP RNAs. In this study, IVT SINE-UPs with nucleotide modifications were stabilized in cells and increased the EGFP level both in a cell-free translation system and in cultured cells. Using cell-free systems, we found that mIVT SINEUPs upregulated EGFP in HeLa cell lysate but not in RRL. Future studies are needed to assess whether chemical nucleotide modifications of IVT SINEUPs contribute not only to RNA stabilization but also to the interaction with SINEUP-binding proteins, which might be necessary for enhancing translation of the target mRNAs.
The modified nucleotides that we used in this study were chosen not only for their ability to stabilize the target transcripts but also for their importance in several regulation systems. The modified nucleotide m 5 C stabilizes tRNA [30], aids accurate processing of ncRNA [31], affects the subcellular localization of transcripts [32], and is involved in the regulatory function of lncRNA [33]. In our current study, although m 5 C-containing mIVT SINEUPs were more stable than IVT SINEUPs (Fig. S2B), m 5 C-did not enhance target protein production (Figs 3B and 5B). Given that RNA modifications change the binding affinities of RBPs [32,34], some positions of m 5 C residues in mIVT SINE-UPs likely diminished their binding to RBPs, which is important for the localization and translational regulation by SINEUPs and warrants further study.
The modified uridine residue Ψ contributes to mRNA stability, intracellular transcript localization [22], and enhancement of mRNA translation through diminishing protein kinase RNA-activated (PKR) activation [23], and is linked to translation accuracy [35].  In addition, RNA duplexes are stabilized when U is replaced with Ψ, leading to the formation of stable Ψ-A, Ψ-G, Ψ-U, and Ψ-C pairs [22]. N 1 mΨ enhances mRNA translation by increasing ribosome density [24] and reduces immunogenicity [25]. Together, these reports suggest that multiple nucleotide modification of RNA has diverse functional possibilities that act through increasing mRNA stability and regulating translation. This knowledge has led to the development of modified mRNA-based approaches for immune cancer therapy and vaccination [36]. In addition to these current nucleic acid-based therapies, SINEUPs have the potential to be used to increase protein production as a biotechnological tool [37], for recombinant antibodies [9,38] and in gene therapy [12], including for diseases that are currently difficult to treat. Adding to current DNA-based gene therapy approaches, RNA-based druggable systems have several advantages, including avoiding the risk of foreign gene integration into the host genome and insertional mutations that may lead to unexpected adverse side effects. In particular, mIVT SINEUP RNAs can be used as an RNA-based drug tool to stimulate only specific target mRNA translation without altering the endogenous mRNA itself, reducing the incidence of unexpected immune responses because of the introduction of modified nucleotides into IVT SINEUPs. Furthermore, mIVT SINEUP RNAs can easily be scaled down to the smallest functional SINEUP RNA, which can then be transported to the target organs with minimal invasive effects to the host. Although how SINEUP RNAs are modified naturally in living cells remains unknown, such modifications, which are missing from IVT transcripts, are likely necessary for SINEUP functions, given that nucleotide modifications can influence protein-RNA interactions and the subcellular distribution of RNAs [39]. The specific modified nucleotides we used in this study are known to alter mRNA secondary structure and thus the half-life and translation efficiency of these transcripts [26]. Ascertaining the importance of the positions of modified nucleotides and improving our understanding of howand which-modified nucleotides contribute to protein upregulation are crucial goals, and continued research into these modifications of SINEUPs and their application as druggable tools is necessary.
In conclusion, we here showed that synthetic, chemically modified IVT SINEUPs have potential as an efficient, protein-producing tool in nucleic acid-based

Conflicts of interest
SZ, SG, and PC are inventors on patent US9353370B2 and related applications in the European Union and Japan, and HT, SZ, SG, and PC are inventors on patent application IT02018000002411 and the related application PCT/IB2019/050914, which is held by SISSA and TranSINE Therapeutics (Cambridge, UK), which was founded by SG and PC and in which HT owns shares. These COIs did not affect the direction and conclusions of this paper.

Author contributions
NT, HT, and PC designed the project with input from SZ and SG; NT performed all experiments and data analysis; and NT, HT, and PC wrote the manuscript with input from SZ and SG.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. The SINEUP targeting mouse Sox9 consists of a BD that overlaps the Sox9 mRNA sequence and an ED containing an inverted SINEB2 sequence from mouse AS-Uchl1 RNA. The SINEUP was cloned into the XhoI and XbaI sites of pCS2+ (A). Underlining highlights BD of Sox9 mRNA; ED is italicized, and restriction sites are in red (XhoI, CTCGAG; XbaI, TCTAGA).   Fold change in the protein levels is shown as means AE SD of at least three independent experiments. ***P < 0.001, ns, not significant (two-tailed Student's t-test). Table S1. FISH probe sequences for SINEUP RNAs. Table S2. FISH probe sequences for EGFP mRNAs.