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piRNAs Interact with Cold-Shock Domain-Containing RNA Binding Proteins and Regulate Neuronal Gene Expression During Differentiation

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Abstract

piRNAs (PIWI-interacting RNAs) are a class of small non-coding RNAs (ncRNAs) abundantly expressed in germline cells and involved in suppressing the transposon activity. Interestingly, recent studies have found piRNA expression in the central nervous system (CNS), yet the underlying biological significance remains largely unknown. In this study, we investigated the expression and function of piRNAs during the retinoic acid (RA)-mediated neuronal differentiation in NT2 cells, a human embryonal carcinoma cell line. We identified a cohort of differentially expressed piRNAs by microarray. Two piRNAs, DQ582359 and DQ596268, were increasingly upregulated during the RA-induced differentiation and involved in regulating the expression of neuronal markers, MAP2 and TUBB3. Furthermore, these piRNAs were found to associate with cold-shock domain (CSD)-containing RNA binding proteins, DIS3, DIS3L2, and YB-1. Markedly, overexpression of these piRNAs further enhanced the protein levels of MAP2 and TUBB3, potentially by downregulating DIS3, DIS3L2, and YB-1. Hence, our study has identified a novel somatic function of piRNAs in regulating neuronal gene expression. The interaction of piRNA with some CSD-containing proteins can be further explored to enhance neuronal differentiation to treat neurodegenerative diseases.

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Acknowledgements

We would like to thank Ms. Michelle Mok and Ms. Wang Xianhui (Department of Biological Sciences, National University of Singapore (NUS)) for their assistance in the mass spectrometry analysis; Dr. Deepan Balakrishnan (Department of Biological Sciences, NUS) for his help in generating the heat map; and Dr. Stepanka Vanacova (Central European Institute of Technology, Masaryk University, Czech Republic) for the kind gift of the DIS3L2 overexpression plasmid. We also want to thank Mr. Sylvester Wong Shu Ming for helping with imaging and bioinformatics support and Mr. Lal Zo Thanga for administrative assistance. The GTEx data described in this manuscript were obtained from the GTEx Portal (https://www.gtexportal.org/home/gene/LINC01015; https://www.gtexportal.org/home/gene/LINC00905), dbGaP accession number phs000424.v8.p2. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.

Funding

This study was supported by the National University of Singapore Start-up Grant R-181–000-155–133, Ministry of Education Tier 1 Grant R-181–000-179–114, and National University Health System Seed Fund R-181–000-192–114. C.S.S. and C.W. were supported by the NUS Postgraduate Scholarships.

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  1. Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Singapore, 117594, Singapore

    Charannya Sozheesvari Subhramanyam, Qiong Cao, Cheng Wang, Zealyn Shi-Lin Heng & Qidong Hu

  2. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Drive, Singapore, 117593, Singapore

    Zhihong Zhou

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  1. Charannya Sozheesvari Subhramanyam
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Contributions

C.S.S. performed most of the experiments in the study. Q.C., C.W., Z.S.L.H., and Z.Z. provided technical assistance in cell culture, Northern blotting, and imaging. Q.H. initiated and managed the project. C.S.S. and Q.H. analyzed the data and wrote the manuscript.

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12035_2021_2678_MOESM1_ESM.pdf

Supplementary file1 Supplementary Fig. 1 Profiling of RA-mediated neuronal gene expression in NT2 cells. qPCR was performed to show the suppression of ESC markers (a) and the induction of neuronal markers (b) at different time points of RA treatment. d: days of RA treatment. Bars: mean · SD; n=3. (c) Western blot was performed to profile the protein levels of ESC and neuronal markers at different time points of RA treatment. (PDF 139 KB)

12035_2021_2678_MOESM2_ESM.pdf

Supplementary file2 Supplementary Fig. 2 Immunofluorescent staining of MAP2 in 15-day RA-treated NT2 cells. NT2 cells were treated with RA for 15 days and then immunostained for MAP2. The cells were counterstained with Hoechst 33342 dye. Scale bar: 5µm. (PDF 96 KB)

12035_2021_2678_MOESM3_ESM.pdf

Supplementary file3 Supplementary Fig. 3 Profiling of piRNA expression in non-treated and RA-treated NT2 cells. (a) Box plot shows the normalized intensities of piRNA array data. (b) Scatter plot shows the differentially expressed piRNAs between indicated samples. (c) Bar graphs illustrate the number of upregulated and downregulated piRNAs from comparisons between non-treated and RA-treated cells as indicated. Ctrl: non-treated cells; 6dRA: 6 days of RA treatment; 15dRA: 15 days of RA treatment; DE: differentially expressed. (PDF 140 KB)

12035_2021_2678_MOESM4_ESM.pdf

Supplementary file4 Supplementary Fig. 4 Interaction of piRNAs with PIWI protein. HA-tagged PIWIL4 protein was overexpressed in 293T cells and piRNA mimics were used to pull down the interacting protein. Western blot with anti-HA antibody shows the association of piR1 and piR2 with PIWIL4. C: control Vector; PL4: HA-tagged PIWIL4 overexpression; piR1: pull-down sample with piR1 probe; piR2: pull-down sample with piR2 probe; piNC: pull-down sample with the scrambled sequence probe. (PDF 94 KB)

12035_2021_2678_MOESM5_ESM.pdf

Supplementary file5 Supplementary Fig. 5 Secondary antibody control for immunofluorescent staining in NT2 cells. The cells were immunostained with anti-Rabbit Alexa 555 secondary antibody with or without pre-labeling with rabbit anti-YB1 antibody, or with anti-mouse Alexa 546 secondary antibody with or without pre-labeling with mouse anti-DIS3 antibody. Scale bar: 20µm. (PDF 109 KB)

12035_2021_2678_MOESM6_ESM.pdf

Supplementary file6 Supplementary Fig. 6 DIS3L2 overexpression affects piRNA-mediated neuronal gene expression. piR1 was concurrently overexpressed along with its interacting partner, DIS3L2 in RA-treated NT2 cells. The expression levels of MAP2 and DIS3L2 were then assessed by Western blot. Ctrl: control Vector; DIS3L2: pEGFP-Flag-HA-DIS3L2 overexpression plasmid; piNC: scrambled sequence. (PDF 93 KB)

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Subhramanyam, C.S., Cao, Q., Wang, C. et al. piRNAs Interact with Cold-Shock Domain-Containing RNA Binding Proteins and Regulate Neuronal Gene Expression During Differentiation. Mol Neurobiol 59, 1285–1300 (2022). https://doi.org/10.1007/s12035-021-02678-2

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