LncRNA Pnky Positively Regulates Neural Stem Cell Migration by Modulating mRNA Splicing and Export of Target Genes

Directed migration of neural stem cells (NSCs) is critical for embryonic neurogenesis and the healing of neurological injuries. The long noncoding RNA (lncRNA) Pnky has been reported to regulate neuronal differentiation of NSCs by interacting with PTBP1. However, its regulatory effect on NSC migration remains to be determined. Herein, we identified that Pnky is also a key regulator of NSC migration in mice, as underscored by the finding that Pnky silencing suppressed but Pnky overexpression promoted the in vitro migration of both C17.2 and NE4C murine NSCs. Additionally, in vivo cell tracking demonstrated that Pnky depletion attenuated but Pnky overexpression facilitated the migration of NE4C cells in the spinal canal after transplantation via injection into the spinal canal. Mechanistically, Pnky regulated the expression of a core set of critical regulators that direct NSC migration, including MMP2, MMP9, Connexin43, Paxillin, AKT, ERK, and P38MAPK. Using catRAPID, a web server for large-scale prediction of protein–RNA interactions, the splicing factors U2AF1 and U2AF1L4, as well as the mRNA export adaptors SARNP, Aly/Ref, and THOC7, were predicted to interact strongly with Pnky. Further investigations using colocalization and RNA immunoprecipitation (RIP) assays confirmed the direct binding of Pnky to U2AF1, SARNP, Aly/Ref, and THOC7. Transcriptomic profiling revealed that as many as 5319 differential splicing events of 3848 genes, which were highly enriched in focal adhesion, PI3K-Akt and MAPK signaling pathways, were affected by Pnky depletion. The predominant subtype of differential splicing by Pnky depletion is intron retention, followed by alternative 5' and 3' splice sites and mutually exclusive exons. Moreover, Pnky knockdown substantially blocked but Pnky overexpression facilitated the export of MMP2, Paxillin, AKT, p38MAPK, and other mRNAs to the cytosol. Collectively, our data showed that through interacting with U2AF1, SARNP, Aly/Ref, and THOC7, Pnky couples and modulates the splicing and export of target mRNAs, which consequently controlling NSC migration. These findings provide a possible theoretical basis of NSC migration regulation.


Introduction
LncRNAs are a class of heterogeneous transcripts that range from 200 nt up to ~ 100,000 nt in length (Constanty and Shkumatava 2021). The mammalian genome encodes many thousands of lncRNAs that have no apparent proteincoding potential, but emerging data indicate that such lncR-NAs can have critical biological functions in development and disease (Chi et al. 2019;Kopp 2019;Wang et al. 2022). Neural stem cells (NSCs) are multipotent cells that have the capacity to self-renew, migrate, and differentiate into multiple lineages, such as the neuronal, astrocytic, and oligodendrocytic lineages (Li et al. 2021a). To date, various lncRNAs have been identified to control NSC behaviors. Jiannan Du and Yuan Li both have contributed equally to this work.
Putative lncRNAs such as Neat1, RMST,LncKdm2b,and EPS have been demonstrated to regulate neuronal differentiation, apoptosis, and neurogenesis by modulating a variety of transcriptional or post-transcriptional processes Ng et al. 2013;Li et al. 2020a;Zhang et al. 2020a, b). However, to date, few lncRNAs have been investigated to determine whether they have potential biological functions in NSC migration.
The lncRNA Pnky, identified by Ramos et al., is an 825 nt, evolutionarily conserved, nuclear-enriched lncRNA that is expressed in NSCs both in vitro and in vivo (Ramos et al. 2015;Cho and Hsieh 2015). Through interacting with PTBP1, the lncRNA Pnky regulates the expression and alternative splicing of a core set of transcripts related to cellular phenotype, which in turn affects neuronal differentiation of embryonic and postnatal NSCs (Ramos et al. 2015;Grammatikakis and Gorospe 2016). Further investigations found that the lncRNA Pnky is a trans-acting regulator of cortical development in vivo (Andersen et al. 2019). However, the functional significance of the lncRNA Pnky in NSC migration has rarely been studied.
Recent studies have reported that the regulation of alternative splicing and mRNA nuclear export patterns, which play a critical role in increasing transcriptomic and proteomic diversity, is generally considered to be involved in every step of nervous system development, including NSC migration (Su et al. 2018;Zheng 2020;Fila et al. 2021). Accumulating evidence has shown that nuclear-enriched lncRNAs may regulate the alternative splicing or mRNA export of target genes. For instance, the lncRNA HOTTIP was identified to facilitate cell migration in osteosarcoma by binding to the splicing regulator PTBP1 (Yao et al. 2021). The lncRNA MALAT1, which is specifically expressed in nuclear speckles, was reported to regulate cell migration and differentiation by controlling RNA splicing and chimeric mRNA export processes Chen et al. 2020). Recently, another lncRNA, NKILA, was found to inhibit breast cancer cell migration by modulating nuclear export via the TREX/TAP pathway (Khan et al. 2021). However, few lncRNAs have been reported to regulate NSC migration by influencing the splicing and mRNA export processes. In our study, a preliminary Pnky-protein interaction analysis provided evidence that Pnky can potentially bind to the splicing factor U2AF1 as well as to some mRNA export adaptors SARNP, Aly/Ref, or THOC7, yet the links among Pnky, mRNA splicing, mRNA export, and NSC migration remain to be elucidated.
In this paper, we aim to investigate the possible role of the lncRNA Pnky in NSC migration, as well as the mechanisms underlying its modulatory effects. Remarkably, we discovered that Pnky can positively regulate the migration of both C17.2 and NE4C cells by regulating the expression of some critical regulators involved in NSC migration, including MMP2, MMP9, Connexin43, Paxillin, PI3K/Akt, p38MAPK, and ERK. Regarding the precise internal mechanism, we found that, on the one hand, the lncRNA Pnky can directly bind to the splicing factor U2AF1, thereby modulating the mRNA splicing process. On the other hand, as an RNA scaffold, Pnky directly recruits SARNP, Aly/Ref, and THOC7 to the transcription-export (TREX) complex and subsequently regulates the export process of target mRNAs. These findings establish a Pnky-regulated mRNA splicing/ export program, providing critical new insight into NSC migration regulation.

3
C17.2 and NE4C cells stably expressing Pnky shRNA were generated by lentiviral transduction. In brief, HEK 293 T cells cultured in 6-cm dishes were transduced with 6 μg of the Pnky shRNA-expressing lentiviral vectors and 6 μg of the packaging vectors PH1 and PH2, using Lipofectamine 3000 (#L3000001, Invitrogen, MA, USA). After 48 h, the lentiviral particles were collected and concentrated. C17.2 and NE4C cells cultured in 12-well plates were infected with the obtained lentiviral particles, and cell lines stably expressing Pnky shRNA were screened from single-cell colonies using 96-well plates as described previously (Peng et al. 2020). Using the same strategy, we generated C17.2 and NE4C cells stably expressing Pnky gene. To obtain control cell lines, lentiviral particles expressing control shRNA were purchased and used for lentiviral transduction directly. An empty vector pLV-U6-CMV-Puro was used for the generation of mock controls.

RNA Isolation and RT-qPCR
Total RNA was extracted with the RNA miniprep kit (#AP-MN-MS-RNA-50, Axygen). Reverse transcription was performed with 0.5 μg of RNA using HiScript III 1st Strand cDNA Synthesis Kit (#R312-01, Vazyme, China) for detecting Pnky expression, or RevertAid First-Strand cDNA Synthesis Kit (#K1622, Thermo Scientific) for determining the expression of related coding genes. Quantitative PCR reactions (three technical replicates per sample) were carried out using FastStart Universal SYBR Green Master (#4,913,914,001, Roche, Germany) and gene-specific primers in a Bio-Rad CFX96 detection system (Bio-Rad, CA, USA). Relative mRNA levels were analyzed with CFX Manager 3.1 software (Bio-Rad) using the 2 −ΔΔCt method. The housekeeping gene (GAPDH) was used for normalization. Differences in gene expression were determined by six independent experiments.

Immunofluorescence Staining
Immunofluorescence staining for Nestin was performed with a standard protocol. Briefly, cells grown on the 14 mm (diameter) coverslips were fixed with 4% paraformaldehyde (PFA, Beyotime, China) for 20 min, followed by permeabilization with 0.2% Triton X-100 for 10 min at room temperature. Samples were then incubated with 10% goat serum for 1 h to block any non-specific interactions. Antibody against Nestin (#ab6142, Abcam, CA, USA; 1:100 dilution) was added to the samples for incubation at 4 °C overnight. After 3 washes with PBS, cells were further incubated with Cy3conjuated secondary antibody (#A0521, Beyotime; 1:500 dilution) for 2 h at room temperature. Nuclei were counterstained with mounting medium (with DAPI; #ab104139, Abcam). Finally, the samples were imaged using an Olympus fluorescence microscope (CKX53, Olympus, Japan). The percentages of Nestin-positive cells were determined by six biological replicates (with three technical replicates each) using Image-Pro Plus 6.0 software.

Cell Proliferation Assays
Cell counting and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assays were performed to determine the cell proliferation rates of the stable cell lines. For cell counting assay, C17.2 and NE4C cells stably expressing Pnky shRNA or Pnky gene, as well as their corresponding control cells, were seeded into 12-well plates in quintuplicate at a concentration of 3*10 4 . Cell samples were collected every 24 h for a total of 5 days after seeding, and differential cell counts were obtained automatically using a Countstar IC1000 cell counter (Countstar, China). The test was independently repeated six times by seeding new batches of cells on new batches of plates.
EdU is a thymidine analogue that can be incorporated into cellular DNA during DNA replication and is usually adopted in the study of cell proliferation . As for EdU incorporation assay, the BeyoClick™ EdU cell proliferation kit with Alexa Fluor 555 (# C0075S, Beyotime) was used according to the manufacturer's instructions. Briefly, cells in the logarithmic phase were seeded into 12-well plates in triplicate at a concentration of 3*10 5 and incubated overnight. The nest day, 10 μM EdU solution was added for 2-h incubation. Subsequently, cells were fixed with 4% PFA for 15 min and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. After washing with PBS, 250 μl of click reaction solution was added and allowed for incubation for 30 min in dark. Finally, nuclei were counterstained with mounting medium (with DAPI). At least 10 fields were selected randomly in each group and photos were taken under the Olympus fluorescence microscope with the magnification of 200 × . The percentages of EdU-positive cells were determined by six independent experiments.

Scratch Wound Migration and Invasion Assay
The effect of the lncRNA Pnky in NSC migration was explored using scratch wound migration and invasion assay. C17.2 and NE4C cells stably expressing Pnky shRNA or Pnky gene, as well as their corresponding control cells, were seeded onto 6-well plates (three technical replicates per cell line) at a concentration of 3*10 6 and cultured to 80% confluences in complete medium. Three parallel scratches were made using sterile pipette tips, and cell debris was washed off using PBS. Then, cells were further incubated in medium with 2% (v/v) FBS for 48 h. The closure of scratch wounds was captured randomly using the Olympus microscope at the designated time points of 0 h, 24 h, and 48 h, respectively. Wound widths were measured and analyzed using ImageJ software. The cell migration status was assessed synthetically based on six independent experiments.

Transwell Migration Assay
Transwell assay was also used to evaluate the migration of the obtained stable cell lines. In brief, 1*10 5 cells were suspended in 200 μl of serum-free medium and transferred to the upper chambers (# 3422, 8.0 μm pores, Corning, USA) that placed into 24-well plates (three chambers per cell line). The bottom chambers were filled with 600 μl of medium containing 10% (v/v) FBS. After incubation for 24 h, the chambers were washed gently with cold PBS and replaced into new 24-well plates. Cells that migrated to the lower surface of the membrane were fixed with 1 ml of methanol for 10 min, followed by staining with 1% crystal violet for 20 min. However, cells that, on the upper membrane surface, were wiped off using fluffy swabs were soaked with PBS. The numbers of the migrated cells were counted manually in 5 ~ 8 randomly selected fields under the Olympus microscope. Images were photographed with the magnification of 200 × . Six biological replicates were performed to evaluate the migration ability of cells.

Intraspinal Cavity Injection, In Vivo Tracking of NSCs, and Hematoxylin-Eosin (H&E) Staining
To explore the function of Pnky in NSC migration in vivo, NE4C cells stably expressing Pnky shRNA or Pnky gene, as well as their corresponding control cells, were fluorescently labeled with the lipophilic tracer DiI (# C1036, Beyotime) in advance. Briefly, cells were seeded into 6-cm dishes and cultured for 12 h. Then, 10 μM DiI was added to the dishes and incubated for 15 min. At the designated time points, cells were washed with PBS 3 times to remove free DiI. Fluorescent imaging was further carried out to confirm the successful labeling of cells.
All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the Medical Research Ethics Committee of Wuhan University of Science and Technology. Female C57BL/6 mice (6-8 weeks old, 18-20 g) were purchased from Hubei Experimental Animal Research Center (Wuhan, China) and randomly and equally divided into four groups (six mice per group). All mice were housed in a specific pathogen-free, temperature-controlled environment on a 12-h light/dark cycle. After adaptive feeding for one week, mice were anesthetized with 5% isoflurane, and the surgical area was sterilized with povidone-iodine. After revealing the spinous process at the lumbar 2-3 (L2-3) level, 20 μL of DiI-labeled cells (suspended in PBS at a concentration of 10 7 cells/mL) was injected into the spinal cord, as described in (Kim et al. 2021). Three days post-cell transplantation, a total of 1.0 cm (0.5 cm of spinal cord on each side of the injection point for Pnky shRNA or ctrl shRNA group) or 2.0 cm (for Pnky overexpression or mock ctrl group) of spinal cord was collected from each mouse and fixed in a 4% paraformaldehyde solution overnight at 4 °C, and the sample was then embedded in tissue freezing medium for cryoprotection. The frozen spinal cord was longitudinally sliced into 20-μm-thick sections using a cryostat microtome (CM1950; Leica Microsystems, Germany). After staining with DAPI, the frozen sections were tile-scan imaged using an Olympus confocal laser scanning microscope (FV3000, Olympus, Japan) at 40 × and 200 × . Cell migration in vivo was assessed by measuring the distance between the most distal DiI-stained cell and the leading edge of the injection site using ImageJ software. H&E staining was also performed to visualize the migration status of the transplanted NSCs using the acquired longitudinal sections of the spinal cord. Images were acquired with an Olympus microscope.

Colocalization of the lncRNA Pnky and Protein
As described elsewhere , wild-type NE4C cells were pre-seeded onto coverslips in triplicate and cultured in static condition for 24 h. For the colocalization of Pnky with the corresponding proteins, cells were first hybridized with Pnky probes conjugated with Cy3 (RiboBio), followed by immunofluorescence staining using antibodies of interest. Briefly, cells were gently rinsed in PBS and fixed in 4% PFA for 10 min at room temperature. Subsequently, cells were permeabilized with 0.5% Triton X-100 for 10 min at 4 °C, washed with PBS for 3 times, and pre-hybridizated in pre-hybridization buffer for 30 min at 37 °C. Then, samples were hybridized overnight using Cy3-labeled Pnky probes at 37 °C in a dark moist chamber. On the second day, after sequential washing with 4SSC, 2SSC, and 1SSC at 42 °C in dark, cells were further fixed in 4% PFA for 5 min and incubated with antibodies against SARNP (#ab225694, 1:200 dilution), Aly/Ref (#ab202894, 1:300 dilution), U2AF1 (#ab172614, 1:300 dilution), THOC7 (#ab155218, 1:200 dilution), and U2AF1L4 (#ab188582, 1:250 dilution) at 4 °C overnight, respectively. On the next day, cells were incubated with specific Alexa Fluor 488-conjugated secondary antibodies (#A0428, #A0423, Beyotime, 1:500 dilution) and counterstained with DAPI. A minimum of 12 fields were selected randomly in each group, and confocal z-section was performed at 1-μm intervals by an Olympus confocal laser scanning microscope using the 100 × oil immersion lens. Six biological replicates were performed to analyze the colocalization of Pnky with the corresponding proteins.

RNA Immunoprecipitation (RIP)
RIP experiments were performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (#17-704, Millipore) according to the manufacturer's instructions. Wild-type NE4C cells were harvested from the plates by scraping, collected, and lysed in complete RIP lysis buffer for 30 min on ice. Magnetic beads were incubated with 5 μg of antibodies specific for SARNP (#ab225694), Aly/Ref (#ab202894), U2AF1 (#ab172614), THOC7 (#ab155218), or U2AF1L4 (#ab188582) or with normal mouse/rabbit IgG (obtained from the above kit) for 40 min at room temperature with rotation. Then, bead-antibody complexes were washed twice with 0.5 mL of RIP wash buffer, and the cell lysates were added to the complexes and incubated at 4 °C overnight. Ten percent of each cell lysate (input) was stored at -80 °C until RNA purification. The next day, with a magnetic separator, the RNAs associated with the corresponding proteins were pulled down. The immunoprecipitated and input samples were then subjected to protease K treatment and heated at 55 °C for 30 min for protein digestion. Subsequently, RNAs were purified with phenol:chloroform:isoamyl alcohol (#6800-OP, EMD Chemical Inc.) and precipitated using absolute ethanol. One microgram of RNA was used for cDNA synthesis using a HiScript III 1st Strand cDNA Synthesis Kit, and enrichment of Pnky was detected using RT-qPCR as described in Sect. 2.3. Normal mouse or rabbit IgG was used as a negative control. Fold enrichment of Pnky was calculated relative to the percentage of input from six independent experiments with three technical replicates each. In addition, parallel samples of RNA and RNA-binding protein complexes were treated with SDS lysis buffer for further Western blot analysis to test the efficiency of immunoprecipitation.

RNA Sequencing and Alternative Splicing Analyses
Total RNA from NE4C cells expressing control shRNA or Pnky shRNA was isolated with an RNeasy Mini Kit (Qiagen, Germany). RNA sequencing (n = 3) was performed by Bioyigene Biotechnology Co., Ltd. (Wuhan, China). Briefly, sequencing libraries were generated using an Illumina TruSeq Kit according to the manufacturer's instructions. Transcriptome RNA-seq was performed on the Illumina HiSeq platform using the standard protocol. RNA-seq reads were then aligned and annotated to the reference mouse genome (mm10) using HISAT2 (Kim et al. 2019). StringTie (Pertea et al. 2015) and DESeq2 software (Love et al. 2014) were used to quantify gene-level expression. Differential alternative splicing in unpaired replicates was assessed with rMATS software (4.0.2) using FDR < 0.05 as the threshold criterion. Sashimi plots were generated to visualize alternative splicing events with significant differences. To identify significantly enriched pathways associated with the differential splicing events, the publicly available web tools KEGG (http:// www. genome. jp/ kegg/), as well as clusterProfiler software, were used with a p value < 0.05.

Cytoplasmic RNA Preparation and mRNA Export Analysis
To quantify the cytoplasmic transcript levels of MMP2, MMP9, Connexin43, Paxillin, p38MAPK, AKT and ERK, stable C17.2 and NE4C cells were generated and processed for the preparation of cytoplasmic RNAs using a Cytoplasmic and Nuclear RNA Purification Kit (#21,000, Norgen Biotek Corp., Canada) as per the manufacturer's instructions. Briefly, the cytoplasmic fractions were isolated by incubating cells in ice-cold Lysis Buffer J for 5 min on ice. Then nuclei were pelleted by centrifugation for 10 min at 4000 × g in a benchtop centrifuge, and supernatant containing the cytoplasmic extracts was collected and processed for binding to the column. Buffer SK and absolute ethanol were added sequentially, and the mixture was loaded onto a spin column for centrifugation at 3500 × g for 1 min. After 3 washes using Wash Solution A, cytoplasmic RNA was eluted using Elution Buffer E.
Total RNA was extracted with an RNA miniprep kit (Axygen) as described in Sect. 2.3. Total and cytoplasmic RNA were reverse transcribed using the RevertAid First-Strand cDNA Synthesis Kit according to the manufacturer's instructions. RT-qPCR analyses of MMP2, MMP9, Con-nexin43, Paxillin, ERK, AKT, p38 MAPK, and 18S rRNA were carried out as described in Sect. 2.3. The ratios of cytoplasmic to total mRNA were normalized to 18S rRNA levels and are presented relative to the ratios in the control samples. mRNA export was assessed synthetically by six independent experiments (with three technical replicates each).

Statistical Analysis
Throughout our research, appropriate sample size was determined based on the effect size, α error (0.05), and the Power (0.95), using the program GPower3.1 as reported previously (Serdar et al. 2021). Statistical analysis was carried out using IBM SPSS Statistics (version 20.0, IBM Corp., Armonk, NY, USA). Accordingly, normality and homogeneity of variance were firstly assessed using Kolmogorov-Smirnov Test and Levene's Test. As for parametric data, Student's t test or one-way ANOVA was used to assess the statistical significance, as appropriate, and results are expressed as the mean ± SD of six independent experiments. If the data were not normally distributed or variance homogeneity was not met, Mann-Whitney U test was performed and the data are displayed as the median and interquartile range. Values of P < 0.05 were considered to indicate a statistically significant difference (**P < 0.01, *P < 0.05). As for the statistical analysis of wound healing assay, repeated-measures ANOVA was carried out using IBM SPSS Statistics. In brief, Mauchly's Tests of sphericity were firstly performed. If P > 0.05, sphericity was met and tests assuming sphericity were used to assess the variances between the pairs. In case P < 0.05, sphericity was violated and Greenhouse-Geisser correction would further come into play. GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA) was used to make the statistical charts.

Blind Study Statement
In all our experimental research, study participants, data collectors, and data analysts are kept unaware of group assignment (control vs. intervention).

The lncRNA Pnky Negatively Regulates the Proliferation of NSCs
To investigate the biological functions of Pnky in murine NSCs, we generated C17.2 and NE4C cell lines stably expressing Pnky shRNA or the Pnky gene, as well as the corresponding control cell lines expressing control shRNA or transduced with empty lentiviral vector (mock control), respectively. The mRNA levels of Pnky were confirmed using RT-qPCR. As shown in Fig. 1a and Table S2, expression of Pnky shRNA in C17.2 and NE4C cells substantially inhibited the expression of Pnky, with knockdown efficiencies of 79.0 ± 8.0% (F(2,15) = 131.6, P < 0.001) and 61.7 ± 15.0% (F(2,15) = 57.35, P < 0.001), respectively, compared to that in the wild-type counterparts. In contrast, in cells stably transduced with the Pnky lentiviral expression vector, Pnky expression was significantly upregulated, with increases of approximately 121.96 ± 22.99-fold (F(2,15) = 165.97, P < 0.001) and 302.60 ± 98.99-fold (F(2,15) = 55.68, P < 0.001) compared with that in wild-type C17.2 and NE4C cells, respectively ( Fig. 1e and Table S3, P < 0.001). However, Pnky expression in control cell lines expressing control shRNA or mock control was not significantly different from that in the counterpart wild-type cells ( Fig. 1a and e). These data indicated that we successfully generated stable cell lines with Pnky knockdown and overexpression.
Next, the effect of Pnky on stem cell maintenance was assessed using stable C17.2 and NE4C cell lines maintained in growth medium. Immunofluorescence staining showed that the differential expression of Pnky did not affect the stem cell fate of C17.2 and NE4C cells under growth conditions, with a percentage of Nestin-positive cells of up to 98% in each stable cell line ( Fig. 1b and f, Tables S4-5), indicating the undifferentiated state of NSCs under our conditions (Engert et al. 2021). Further investigations of the role of Pnky in cell proliferation were also carried out using cell counting and EdU incorporation assays. The results revealed that silencing of Pnky promoted the proliferation of both C17.2 and NE4C cells, as evidenced by the higher cell proliferation rates and numbers of EdU-positive cells in both the C17.2 and NE4C Pnky shRNA groups, with increases in both of these parameters of ~ 2.32-fold in C17.2 cells and 2.02-fold in NE4C cells vs. the wild-type counterparts ( Fig. 1c and d, Tables S6-7). In contrast, Pnky overexpression decreased cell proliferation ( Fig. 1g and h). However, cell fate and proliferation in control cell lines expressing control shRNA or mock control showed no apparent differences compared with the corresponding wild-type cell lines ( Fig. 1 b-d and f-h, Tables S 4-7).

Pnky Depletion Attenuates But Pnky Overexpression Facilitates the Migration of NSCs
To preliminarily explore the possible role of Pnky in NSC migration in vitro, scratch wound healing and Transwell assays were performed using stable C17.2 and NE4C cell lines. The Transwell migration assay showed that lncRNA Pnky inhibition markedly impaired the migration of both C17.2 and NE4C cells, with migration rates of 27.97 ± 2.5% (t(10) = 23.71, P < 0.001) and 38.98 ± 5.49% (t(10) = 13.54, P < 0.001), respectively, compared to those in the corresponding C17.2 and NE4C control cells expressing control shRNA ( Fig. 2a and Tables S8-9). In addition, both the C17.2 and NE4C Pnky shRNA groups exhibited slower closure of scratch wounds, as seen by the increased wound width percentages in comparison with those in the control groups at 24 h and 48 h ( Fig. 2b and Tables S10-11, P < 0.001). Immunofluorescence detection of Nestin (+ , red) in the above stable cell lines with Pnky silencing. Nuclei were labeled (blue) with DAPI. The percentages of Nestin-positive cells in each group were determined using Image-Pro Plus software. c, d Pnky knockdown promoted the proliferation of C17.2 and NE4C cells. Cell proliferation assays were performed using the cell counting (c) or an EdU incorporation assay (d). e Analysis of Pnky expression in the C17.2 and NE4C cell lines stably expressing the Pnky gene or mock control, compared with the corresponding wild-type cell lines. f Immunofluorescence detection of Nestin (+ , red) in the above-described stable cell lines with Pnky overexpression. Nuclei were labeled (blue) with DAPI. The percentages of Nestin-positive cells in each group were determined using Image-Pro Plus software. g, h Pnky overexpression decreased the proliferation of C17.2 and NE4C cells. Cell proliferation assays were performed using the cell counting (g) or an EdU incorporation assay (H). The data are displayed as the mean ± SD of six independent experiments. **P < 0.01. P values were calculated by one-way ANOVA or Student's t test 1 3 We also evaluated the differences in the migration ability between the Pnky overexpression group and the mock control group (transduced with empty lentiviral vector). The Transwell assay data showed that the number of cells that migrated to the lower surface of the chamber membrane was significantly increased for Pnky-overexpressing C17.2 and NE4C cells, with migration rates of up to 158.22 ± 21.75% (t(10) = -5.93, P < 0.001) and 203.20 ± 17.85% (t(10) = -10.26, P < 0.001), respectively, compared to those of the corresponding mock control cells ( Fig. 2c and Tables S12-13). The wound widths were also evidently decreased in the Pnky overexpression groups relative to the mock control groups ( Fig. 2d and Tables S14-15, P < 0.001).
To further explore the function of Pnky in NSC migration in vivo, NE4C cell line, which shows stronger cell viability and better stability, is used for in vivo cell tracking. In detail, stable NE4C cells were prelabeled with DiI and transplanted into mice (6 animals per group) via injection into the spinal canal. Cell migration was tracked on Day 3 post-cell transplantation. As shown in Fig. 2e and Table S16, transplanted NE4C cells expressing control shRNA showed extensive migration along the spinal canal, with an average migration distance from the injection site (IS) to the final position (FP) of 423.83 ± 65.32 μm. In contrast, NE4C cells with Pnky depletion exhibited markedly impaired migration after transplantation into the spinal canal, with an average migration distance of as low as 250.56 ± 42.66 μm. Additionally, in vivo cell tracking also demonstrated that Pnky overexpression facilitated the migration of NE4C cells in the spinal canal, with the migration distance up to 908.00 ± 103.64 μm in Pnky-overexpressing group (Fig. 2g and Table S17). H&E staining of spinal cord samples displayed that the structure of spinal cords was intact without visible local scars and necrotic cavitation (Fig. 2f and h). Meanwhile, no evidence of neoplasm formation and inflammatory cell infiltration were visualized in either group of the spinal cords post-cell transplantation ( Fig. 2f and h), which demonstrated that the initial transplantation of NE4C cells in the spinal canal did not lead to any damage to the spinal cord. Collectively, these data suggest that Pnky depletion attenuates but Pnky overexpression facilitates the migration of NSCs both in vitro and in vivo.

Pnky Regulates the Expression of Some Critical Regulators and the Activity of Signaling Pathways That Direct NSC Migration
NSC migration is a multistep process that includes the extension of lamellar pseudopodia, formation of new focal contacts, forward contraction of the cell body, and dissociation of the cell tail (Seetharaman and Etienne-Manneville 2020), and many critical regulators are involved in this process. For example, MMP2 and MMP9 are two key metalloproteinases that participate in extracellular matrix degradation, thus facilitating cell migration . Paxillin, the focal adhesion adaptor protein, functions to recruit structural and signaling molecules involved in cell movement and migration (Rajah et al. 2019). Connexin 43, which forms gap junction channels, exerts crucial effects on cellular functions such as proliferation and migration (Zhang et al. 2018).
To investigate whether Pnky affects the above key regulators, Western blot and RT-qPCR analyses were adopted to test their expression in stable C17.2 and NE4C cell lines. As expected, Pnky depletion decreased the expression of MMP2, MMP9, Connexin 43, and Paxillin at both the mRNA and protein levels ( Fig. 3a-b and Tables S18-19, P < 0.001). In contrast, as shown in Fig. 3c-d and Tables S20-21, Pnky overexpression enhanced the expression of these regulators in C17.2 and NE4C cells (P < 0.001). These data indicate that Pnky positively regulates NSC migration by influencing the expression of some critical regulators.
The PI3K/Akt, p38MAPK, and Raf/MEK/ERK signaling pathways have always been a focus of interest due to their roles in cell proliferation, migration, and metabolism (Han et al. 2016;Hirata and Kiyokawa 2019;Shahcheraghi et al. 2020). They are highly involved in the modulation of several downstream effectors, such as MMP2 and Paxillin, and thereby affect cell movement (Cui et al. 2016;Sánchez-Martín et al. 2019). Herein, Western blot analysis was used to identify the pathways involved in the modulatory effect of Pnky on NSC migration. Densitometric analysis and quantification of protein levels were carried out using ImageJ software. The data showed that Pnky knockdown inhibited the expression and phosphorylation of p38MAPK, ERK, and AKT in both C17.2 and NE4C cells, suggesting the suppression of p38MAPK, Raf/MEK/ERK, and PI3K/Akt pathways (Fig. 3 e-f, Tables S22-23), whereas Pnky overexpression activated these pathways (Fig. 3 g-h, Tables S24-25). Overall, this evidence indicates that Pnky regulates these signaling pathways (p38MAPK, Raf/MEK/ERK, and PI3K/Akt) The statistical data in a-e and g are expressed as the mean ± SD of six independent experiments. **, P < 0.01. Accordingly, the P values in a, c, e, and g were calculated by Student's t test. The P values in b and d were calculated by repeated-measures ANOVA. The Mauchly's test of sphericity on the data in b and d suggested that the assumption of sphericity was met (P = 0.43, P = 0.18, P = 0.43 and P = 0.74, respectively), and tests assuming sphericity were used to assess the variances between the pairs ◂ and the key downstream effectors (MMP2, MMP9, Paxillin, and Connexin 43), which in turn directs NSC migration.

Pnky Could Potentially Bind to Several Factors Involved in mRNA Splicing and Export Processes
LncRNAs generally associate with specific sets of RNAbinding proteins (RBPs) to form functional ribonucleoprotein (RNP) complexes, thereby modulating gene transcription, pre-mRNA processing, mature mRNA export, mRNA degradation, protein translation, and ubiquitination (Bridges et al. 2021). To uncover the possible mechanism by which Pnky regulates the expression of the key regulators investigated in Sect. 3.4., we screened for potential proteins that may interact with Pnky using the online tool catRAPID (http:// servi ce. tarta glial ab. com/ email_ redir/ 191801/ 3e2b2 03d12), and the top hits are listed in Table 1. It has been reported that Pnky is a nuclear-enriched lncRNA that functions as a trans-acting regulator (Ramos et al. 2015). On the basis of the interaction strength, protein localization, and Z scores, we found that SARNP, Aly/Ref, U2AF1, U2AF1L4, and THOC7 had high probabilities of directly interacting with Pnky. Interestingly, among these proteins, SARNP, Aly/ Ref, and THOC7 are core components of the TREX complex, which specifically associates with spliced mRNA and functions in mRNA export to the cytoplasm (Heath et al. 2016). U2AF1 is one component of the pre-mRNA splicing machinery and mainly functions to recruit the snRNP U2 to 3' splice sites of pre-mRNAs, thus playing a critical role in both constitutive and enhancer-dependent splicing (Palangat et al. 2019). U2AF1L4, also called U2AF26, is another splicing factor that can functionally substitute for U2AF1 (Preußner et al. 2014). Accordingly, we hypothesized that Pnky might regulate the expression of a core set of genes related to NSC migration by modulating mRNA splicing and export processes.

Pnky Modulates the Cellular mRNA Splicing Landscape by Directly Binding to the Splicing Factor U2AF1
To substantiate our hypothesis, we first determined the specificity of the primary antibodies (anti-U2AF1 and anti-U2AF1L4) using a knockdown validation approach (Fig. S9). Subsequently, colocalization analysis of Pnky and U2AF1 (or U2AF1L4) was carried out using FISH and immunofluorescence costaining techniques. Confocal z-section images showed that the lncRNA Pnky was partially colocalized with U2AF1 or U2AF1L4 in the nucleus of NE4C cells ( Fig. 4a and b). Furthermore, a RIP assay was carried out to examine the direct interactions between these lncRNA-protein pairs. As expected, Pnky was preferentially enriched (28.64 ± 4.86%) on the U2AF1 antibodylinked beads compared to the control IgG-linked beads (1.42 ± 0.54%) ( Fig. 4c and Table S26, P < 0.001), suggesting that Pnky can directly bind to the splicing factor U2AF1. Unexpectedly, Pnky RNA enrichment was very low in the U2AF1L4 immunoprecipitates ( Fig. 4d and Table S27, P = 0.24), indicating the lower affinity of Pnky for U2AF1L4. This may be due to the functional substitution of U2AF1L4 for U2AF1 and the tendency of Pnky to interact preferentially with U2AF1. Moreover, the efficient immunoprecipitation of U2AF1 and U2AF1L4 proteins from NE4C cell extracts was confirmed using Western blot analysis ( Fig. 4c and d, right panels).
Given that Pnky interactor U2AF1 plays a functional role in RNA splicing, we next assessed the alternative splicing of specific pre-mRNAs that might underlie Pnky's physiological role in NSC migration. RNA sequencing analysis of Pnky-depleted NE4C cells revealed an aberrant RNA splicing pattern of 5319 differential splicing events (3848 genes, FDR < 0.05), with 1764 for retained introns (RI), 1489 for alternative 5' splice sites (A5SS), 1444 for alternative 3' splice sites (A3SS), and 622 for mutually exclusive exons (MXE) (Fig. 4e, Table S28). Interestingly, the differentially spliced transcripts associated with Pnky depletion are highly enriched in KEGG pathways including spliceosome, focal adhesion, PI3K-Akt and MAPK signaling pathways, which are closely related to migration regulation (Fig. 4f). Using sashimi plots, we visualized the differential splicing patterns along genomic regions of representative genes with retained introns (Sat1), alternative 5' splice sites (Renbp), alternative 3' splice sites (Chka), or mutually exclusive exons (Cpsf7) (Fig. S11). These findings suggest that Pnky expression changes induce a concerted reprogramming of the splicing pattern of transcripts that crucial for NSC migration.

(c) and NE4C cells (d).
Left panel: RT-qPCR analysis; Right panel: Western blot analysis. e-f Pnky knockdown decreased the expression and phosphorylation of ERK, AKT, and p38MAPK in both C17.2 (e) and NE4C cells (f). g-h Pnky overexpression increased the expression and phosphorylation of ERK, AKT, and p38MAPK in both C17.2 (g) and NE4C cells (h). The corresponding densitometric analysis of these signaling mediators is shown in the right panels of e-h. All values were normalized to the GAPDH signal. Accordingly, the data are displayed as the mean ± SD or median and interquartile range of six independent experiments. **, P < 0.01. P values were calculated by one-way ANOVA or the Mann-Whitney U test. Full Western blots with markers and the corresponding loading controls can be found in Figs. S1-S8. ERK and p-ERK, AKT and p-AKT, and p38MAPK and p-p38MAPK were assessed on the same membranes ◂ Overall, this scientific evidence indicated that by directly binding to the splicing factor U2AF1, the lncRNA Pnky modulates the splicing of mRNAs encoding proteins that direct NSC migration.

Pnky Binds Directly to SARNP, Aly/Ref and THOC7, and Consequently Facilitates the Export of Target mRNAs
The above bioinformatics prediction indicated that the lncRNA Pnky can also potentially bind to SARNP, Aly/ Ref and THOC7. SARNP participates in mRNA splicing and export through binding to UAP56, an RNA helicase component of the TREX complex which is thought to link mRNA transcription, processing, and nuclear export (Kang et al. 2020). Aly/Ref is an export adapter that mediates the recruitment of the TREX complex and is involved in the nuclear export of spliced mRNAs ). THOC7 usually functions as one component of the THO subcomplex, a constituent of the TREX complex, and is required for efficient export of polyadenylated RNA . To verify the bioinformatics predictions, we further studied the colocalization and direct-binding between Pnky and SARNP (or Aly/Ref or THOC7). As above, we first determined the specificity of the corresponding primary antibodies (anti-SARNP, anti-Aly/Ref and anti-THOC7) using a knockdown validation approach (Fig. S9) and then performed colocalization analysis of Pnky and SARNP (or Aly/Ref or THOC7). Notably, the lncRNA Pnky was strongly colocalized with SARNP, Aly/Ref or THOC7 in the nucleus of NE4C cells, as clearly shown in the z-stack series and z-section images in Fig. 5a-c. In addition, immunoprecipitation of Pnky further showed that the anti-SARNP, anti-Aly/Ref and anti-THOC7 antibodies retrieved significant amounts of endogenous Pnky RNA, with ~ 27.88-, 7.92-and 5.04-fold enrichment vs. control IgG (Fig. 5d-f and Tables S29-31, P < 0.001), confirming the direct binding of Pnky to SARNP, Aly/Ref and THOC7. In addition, the successful immunoprecipitation of SARNP, Aly/Ref and THOC7 proteins was confirmed using Western blot analysis (Fig. 5d-f).
SARNP, Aly/Ref and THOC7 are key components of the TREX complex, which plays a vital role in the effective export of mRNAs (Kumar et al. 2020). We next sought to examine how the nuclear export of mRNAs is affected by Pnky. To this end, the cytoplasmic mRNA abundance was normalized to the whole-cell transcript level to identify changes in export occurring independent of other alterations in gene expression. To validate our experimental system and data analysis, cell fractionation was carried out, and the subcellular distribution of the indicated mRNAs was assessed using RT-qPCR. Notably, the ratios of all cytoplasmic mRNAs that we examined, including MMP2, MMP9, Connexin43, Paxillin, ERK, AKT and P38MAPK, were reduced in C17.2 and NE4C cells stably expressing Pnky shRNA compared with the corresponding control cells (Fig. 5g and Tables S32-33). In addition, we observed significant increases in the cytoplasmic mRNA ratio for almost all the tested genes (except for ERK) in both C17.2 and NE4C cells with Pnky overexpression (Fig. 5h and Tables S34-35). These data suggested that the lncRNA Pnky exerts a dominant positive effect on the export of mRNA transcripts that direct NSC migration.
It has been reported that the TREX complex is a dynamic structure that is composed at a minimum of Aly/Ref, UAP56, SARNP, CHTOP and the THO subcomplex (which contains THOC1, THOC2, THOC5, THOC6 and THOC7) (Kumar to the TREX complex, thus facilitating the export of target mRNAs, including those of MMP2, MMP9, Connexin43, Paxillin, p38MAPK and AKT, which subsequently modulate NSC migration. Based on these results, we propose an integrated regulatory model of the mechanism by which the lncRNA Pnky The results are expressed as the median and interquartile range of six independent experiments. The individual data points indicate the fold enrichment of Pnky immunoprecipitated by the U2AF1L4-specific antibody or IgG (P = 0.24 vs. IgG, Mann-Whitney U test). Right panel: Representative immunoblots of U2AF1L4 pulled down using the anti-U2AF1L4 antibody. Full Western blots with markers can be found in Fig. S10. e rMATS analysis for differential alternative splicing events in NE4C cells bearing Pnky shRNA or control shRNA. Different types of alternative splicing events were quantified. f Dot plot of significantly enriched pathways using functional enrichment analyses with differential splicing events-related genes affects NSC migration. As shown in Fig. 6, in the nucleus of NSCs, the lncRNA Pnky can directly bind to the splicing factor U2AF1 and consequently reprogram the splicing patterns of target pre-mRNAs by RI, A5SS, A3SS and MXE. In addition, Pnky can also act as an RNA scaffold to recruit SARNP, Aly/Ref and THOC7 to the TREX complex, thus boosting mRNA export to the cytoplasm. Through coordination of mRNA splicing and export processes, the expression of downstream targets, including MMP2, MMP9, Connexin 43, Paxillin, p38MAPK, AKT and ERK, is enhanced, which eventually facilitates NSC migration. Moreover, the phosphorylation of p38MAPK, ERK and AKT is positively regulated in migration, resulting in activated transcription of target genes. Consequently, a positive feedback loop is formed in the Pnky-directed NSC migration process. The ratios of cytoplasmic to total mRNA were normalized to the amount of 18S rRNA and are presented relative to the ratios in the control samples. The data are shown as the mean ± SD or median and interquartile range of six independent experiments. *P < 0.05; **P < 0.01. P values were calculated by one-way ANOVA or Mann-Whitney U test

Discussion
Migration is one of the most crucial characteristics of NSCs, and revealing the regulatory mechanism of NSC migration is the key to revealing the process of neurogenesis and provides new insights for the treatment of neurodevelopmental or neurodegenerative diseases. In recent years, the fundamental roles of lncRNAs in the central nervous system (CNS) have become increasingly evident, and an increasing number of lncRNAs have emerged as critical regulators of neurodevelopmental processes, such as self-renewal and proliferation (Fan et al. 2020;Zhao et al. 2020), neural differentiation (Biscarini et al. 2018;Rea et al. 2020;Winzi et al. 2018;Zhao et al. 2020) and gliogenesis (Bian et al. 2019;Li et al. 2018;Xia et al. 2020). However, the lncRNAs that modulate NSC migration are less well understood.
Previously, the lncRNA Pnky was known for its important role in regulating neuronal differentiation of embryonic and postnatal NSCs (Ramos et al. 2015). Through interacting with PTBP1, the lncRNA Pnky regulates the expression and alternative splicing of a core set of transcripts related to the NSC-neuron transition (Grammatikakis and Gorospe 2016;Ramos et al. 2015). Herein, we identified that the lncRNA Pnky positively regulates NSC migration, as underscored by the finding that silencing of Pnky suppressed but overexpression of Pnky promoted the migration of both C17.2 and NE4C cells (Fig. 2). Moreover, we discovered that Pnky negatively regulated the proliferation of NSCs (Fig. 1), which excluded the possibility that the observed changes in NSC migration were due to cell proliferation and further confirmed the regulatory effect of Pnky on NSC migration.
It is well established that complex gene interactions and molecular networks participate in the NSC migration process (De Gioia et al. 2020). Metalloproteinases and cell adhesion molecules, which facilitate the degradation of old focal adhesions and the formation of new ones, are reported to play pivotal roles in the migration process (Alaseem et al. 2019). Moreover, the PI3K/Akt, p38MAPK and Raf/MEK/  Zhang et al. 2015). Under our conditions, the metalloproteinases MMP2 and MMP9, as well as the focal adhesion adaptor protein Paxillin, were strikingly downregulated at both the mRNA and protein levels, as observed in Pnkysilenced C17.2 and NE4C cells ( Fig. 3a and b). In addition, acute knockdown of Pnky led to significant downregulation of p38MAPK, ERK and AKT phosphorylation in both C17.2 and NE4C cells, while Pnky overexpression generally promoted the phosphorylation of these proteins (Fig. 3 e-h). Interestingly, however, the differential expression of Pnky also showed an impact on total p38MAPK, ERK and AKT expression, suggesting that Pnky indeed regulates the expression of a core set of transcripts that direct NSC migration.
The biological functions of lncRNAs are usually closely related to their intracellular localization (Chen 2016). Pnky is reported to be a predominantly nuclear and evolutionarily conserved lncRNA that is expressed only in neural tissues (Cho et al. 2015;Ramos et al. 2015). Consistent with those findings, our results also showed the nuclear localization of Pnky (Figs. 4 and 5). Models suggest that nuclear lncR-NAs are not limited to a defined set of functions but can regulate diverse activities ranging from structural functions such as chromatin organization or structural scaffolding of nuclear domains to regulatory functions such as transcriptional regulation and pre-mRNA splicing, among others Statello et al. 2021). Moreover, accumulating evidence indicates that nuclear lncRNAs often interact with various nuclear RBPs to perform their regulatory functions. To explore how Pnky affects the expression of MMP2, MMP9, Connexin 43 and Paxillin, which in turn modulate NEC migration, the interactions between Pnky and RBPs were predicted using the catRAPID algorithm. Strikingly, we found that SARNP, Aly/Ref, U2AF1, U2AF1L4 and THOC7, which commonly participate in mRNA splicing and export processes, were predicted to directly interact with Pnky (Table 1), implying that Pnky might play an active role in modulating mRNA splicing and export processes.
Regarding pre-mRNA splicing, which is thought to be a mechanism of posttranscriptional regulation of gene expression, the spliceosome is the executioner that is responsible for removing introns from pre-mRNAs in all eukaryotes (Yan et al. 2019). The spliceosome is composed of at least five small nuclear ribonucleoproteins (snRNPs; U1, U2, U4/U6, and U5) that recognize intronic splice sites by base pairing, organize the assembly of protein splicing factors, and catalyze cleavage and ligation reactions (Shi 2017; van der Feltz and Hoskins 2019). U2AF1 is the small subunit of the U2 auxiliary factor (U2AF) that is a constituent of the snRNP U2, which is primarily responsible for 3' splice site selection during splicing (Kováčová et al. 2020).
Accumulating evidence has shown that U2AF1 mutation causes altered pre-mRNA binding and splicing kinetics in a variety of cell types, such as hematological malignancies and lung adenocarcinomas (Carruale et al. 2019;Esfahani et al. 2019;Palangat et al. 2019). In a previous study, Pnky was reported to regulate alternative splicing by interacting with PTBP1 (Ramos et al. 2015). However, in our study, further validation of the Pnky interactome confirmed that in addition to interacting with its known binding partner PTBP1, Pnky also interacts with U2AF1, as evidenced by the colocalization and RIP assay results (Fig. 4a and c). Moreover, using RNA-seq, we identified as many as 5319 differential splicing events of 3848 genes in NE4C cells transduced with Pnky shRNA or control shRNA (Fig. 4e), which further confirmed that Pnky indeed regulate the splicing of target mRNAs through interacting with U2AF1. Surprisingly, it seems unlikely that Pnky can directly bind to U2AF1L4, another splicing factor that functionally substitutes for U2AF1, as evidenced in Fig. 4b and d. Most likely, the low affinity of Pnky for U2AF1L4 may be attributed to the low expression and functional substitution of U2AF1L4.
The processes of mRNA splicing and translation are coupled by the export of mature mRNAs from the nucleus to the cytoplasm. A large proportion of mRNA export is specifically controlled by the dynamic TREX complex, which contains multiple proteins, including Aly/Ref, UAP56, SARNP, CHTOP and the THO subcomplex (containing THOC1, THOC2, THOC7, etc.) (Williams et al. 2018). During splicing, UAP56 is first recruited to a messenger ribonucleoprotein (mRNP), where it is involved in ATPdependent recruitment of Aly/Ref, SARNP and the THO subcomplex to the mRNP, thus forming the TREX complex (Williams et al. 2018). CHTOP binds to UAP56 in a mutually exclusive manner with Aly/ref (Chang et al. 2013). Abnormal expression of Aly/Ref, SARNP and THO subunits is found in cells of multiple cancers, such as prostate cancer, hepatoma, pancreatic carcinoma and myeloid leukemia, and can directly affect their migration and invasion activities of these cells by regulating mRNA export (Borden 2020;Tran et al. 2016). Furthermore, many intracellular factors have been reported to affect the migration behavior of cells by modulating mRNA export. For instance, NSun2 promotes cell migration by mediating the methylation of ATX mRNA, which can facilitate its export in an Aly/Ref-dependent manner (Xu et al. 2020). The lncRNA MALAT1 controls the chimeric mRNA export process and regulates myeloid progenitor cell differentiation and migration during malignant hematopoiesis (Chen et al. 2020).
Strikingly, in our present study, we confirmed the direct binding of Pnky to SARNP, Aly/Ref and THOC7, manifested as high enrichment of Pnky in the RIP assay ( Fig. 5 d-f). In addition, the cytoplasmic distribution of all mRNAs we examined, including MMP2, MMP9, Connexin43, Paxillin, AKT, ERK and P38MAPK, which are well-established regulators participating in cell migration, was reduced in C17.2 and NE4C cells with Pnky depletion (Fig. 5g). Consistent with these findings, Pnky overexpression increased the cytoplasmic ratios of the tested genes (except for ERK) (Fig. 5h). Therefore, it is not extraordinary to conclude that Pnky modulates the mRNA export process of a core set of factors involved in NSC migration by binding to SARNP, Aly/Ref and THOC7. Moreover, effective mRNA export is an essential prerequisite of cytoplasmic protein expression, and blocking of mRNA export results in differential alterations in protein expression and cell defects (Okamura et al. 2018). Consistent with this observation, the protein expression levels of MMP2, MMP9, Connexin43, Paxillin, AKT, ERK and P38MAPK, were also found to be altered in NSCs with Pnky silencing or overexpression (Fig. 3). This evidence indirectly supports the hypothesis that Pnky may regulate mRNA export by interacting with SARNP, Aly/Ref and THOC7.
Furthermore, emerging studies have shown that the TREX complex is recruited to mRNAs predominantly by the splicing machinery (Williams et al. 2018). More interestingly, our results showed that the lncRNA Pnky can directly bind to both a splicing factor (U2AF1) and mRNA export adaptors (SARNP, Aly/Ref and THOC7), thus coupling the mRNA splicing and export processes of target genes (including MMP2, MMP9, Connexin43, Paxillin, AKT, ERK and P38MAPK). Thus, it is reasonable to speculate that initially, as a splicing machinery regulator, the lncRNA Pnky modulates the pre-mRNA splicing of target genes by interacting with U2AF1. Subsequently, Pnky is more likely to act as an RNA scaffold that recruits SARNP, Aly/Ref and THOC7 to the TREX complex, thus facilitating the export of crucial mRNAs involved in NSC migration.
Taken together, the results of this study provide new insight into the contribution of the lncRNA Pnky to NSC behaviors. The functions of Pnky in NSC migration appear to be mediated by coordinating the mRNA splicing and export processes of target genes. Further study is needed to address the details. Once a greater understanding of how these scaffolding complexes are assembled and regulated is achieved, it will be possible to design strategies to selectively utilize specific signaling components to redirect cellular behaviors.
Author Contributions JND and YL designed the study, performed the research, and analyzed the data. YTS, WQZ, and JLZ helped finish some part of the study. CZ and JW helped in material preparation and data analysis. WSD participated in study design and modified the manuscript. SSZ contributed to study design, data collection and analysis, funding acquisition, and preparation of the manuscript. All authors read and approved the final manuscript.