Small Nucleolar RNA Sf-15 Regulates Proliferation and Apoptosis Of
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Wu et al. BMC Molecular Biol (2019) 20:12 https://doi.org/10.1186/s12867-019-0128-9 BMC Molecular Biology RESEARCH ARTICLE Open Access Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells Bo Wu†, Lei Huang†, Wujie Qiu, Xiao Liu, Yawen Shen, Yiping Lu, Zonglin Yang, Xinmei Li, Bin Cui, Shidong Xu, Huili Qiao, Reng Qiu, Lunguang Yao, Yunchao Kan* and Dandan Li* Abstract Background: Small nucleolar RNAs (snoRNAs) function in guiding 2′-O-methylation and pseudouridylation of riboso- mal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. Results: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. fru- giperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further 2 analysis showed that a Ca +-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was signifcantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. Conclusions: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expres- 2 sion of Ca +-induced cell death pathway gene Cn in Sf9 cells. This is the frst time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death. Keywords: snoRNA, Sf-15, Proliferation, Transcriptome, Sf9 cells Background fragments [3–12] and playing regulatory roles in tran- Small nucleolar RNAs (snoRNAs) are one of the scriptional or post-transcriptional levels in human dis- most abundant non-coding RNA species of 60–300 eases [13–24]. For example, the brain-specifc snoRNA, nucleotides (nt) length and function in guiding HBII-52, is involved in the regulation of RNA edit- 2′-O-methylation (mediated by C/D box snoRNAs) and ing and pre-mRNA splicing of serotonin receptor 2C pseudouridylation (by H/ACA box snoRNAs) in ribo- (5-HT2CR) through an 18 nt complementarity to the somal RNAs (rRNAs), small nuclear RNAs (snRNAs), 5-HT2CR pre-mRNA, and plays important roles in tRNAs, and mRNAs [1, 2]. However, in recent years, a Prader-Willi syndrome [17–19, 25–28]. Furthermore, variety of snoRNAs have been found to display novel accumulating evidence suggests that dysregulation of functions, such as processing molecules into smaller snoRNAs contribute to tumorigenesis [20, 29–32]. For example, accumulation of gas5-generated snoRNAs was related to growth arrest of breast cancer cells [12, *Correspondence: [email protected]; [email protected] 33]. Inhibition of tumor-abundant SNORD78 sup- †Bo Wu and Lei Huang contributed equally to this work China-UK-NYNU-RRes Joint Laboratory of Insect Biology, Henan Key pressed the proliferation of non-small cell lung cancer Laboratory of Insect Biology in Funiu Mountain, Nanyang Normal (NSCLC) cells via inducing G0/G1 cell cycle arrest and University, 1638 Wolong Road, Nanyang 473061, Henan, China apoptosis, while SNORD78 overexpression promoted © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu et al. BMC Molecular Biol (2019) 20:12 Page 2 of 11 cell proliferation [15]. Additionally, SNORD113-1 was participate in cell growth through Ca2+-induced cell found to inactivate the phosphorylation of ERK1/2 and death pathway, which may provide new clues for under- SMAD2/3 in MAPK/ERK and TGF-β pathways and standing the function of snoRNAs. suppress tumorigenesis in hepatocellular carcinoma [34], which indicated that the biological function of Results snoRNAs are various and elusive. Detect the presence of Bm‑15 in Sf9 cell SnoRNAs in lepidopterous insects of silkworm Bom- In our previous studies, we found that a C/D box snoRNA byx mori have been studied by Li et al. [2], but their func- Bm-15 can interact with a Notch receptor gene in B. mori. tional roles have not been fully elucidated. In a previous To further study the function of Bm-15, we cloned its study, we found a C/D box snoRNA, Bm-15, can interact homolog in S. frugiperda Sf9 cells (whose transfection with a Notch receptor gene in vitro [2]. To further study efciency is much higher than cell lines of silkworm). We the function of this snoRNA, we cloned its homolog from found that Bm-15 was conserved between B. mori and Spodoptera frugiperda Sf9 cells, we found that Bm-15 S. frugiperda, with sequence identity of 97% and simi- was highly conserved between B. mori and S. frugiperda. larly secondary structure (Fig. 1a, b). Eukaryotic C/D box Further analysis showed that suppression of Sf-15 inhib- snoRNAs guide 2′-O-methylation of rRNAs, snRNAs, ited cell growth and promoted cell apoptosis. Transcrip- and tRNAs through C/D box small nucleolar ribonu- tome analysis showed that a serine/threonine-protein cleoprotein particles (C/D box snoRNPs), which involves phosphatase gene Cn was a potential target of Sf-15. the core proteins of Snu13p/15.5 K, Nop58p/NOP58, Tis is the frst report that a lepidopterous snoRNA can Nop56p/NOP56, and Nop1p/Fibrillarin. To determine Fig. 1 Characterization of Sf-15 in Sf9 cells. a Sequence identity of Bm-15 between B. mori and S. frigiperda. b Secondary structure of Sf-15 in S. frigiperda. c Abundance of Sf-15 that was pulled down by the antibody of Fibrillarin. M, 100 bp DNA ladders. 1–3 represented PCR products that were amplifed from RNAs being pulled down by anti-Fibrillarin, serum and input, respectively. d Expression of Sf-15 in nucleus and cytoplasm of Sf9 cells. N Nucleus, C cytoplasm. e Relative expression of Sf-15 in nucleus and cytoplasm by quantitative real-time PCR. Results were calculated by relative Ct of diferent genes. f Fluorescent in situ hybridization of Sf-15. Phase represented cells observed under white light. U3 snoRNA was used as a nucleolar marker and was visualized by employing a 5′-FITC-labeled antisense oligonucleotide. The in situ hybridization probes of Sf-15 were labeled with Cy3 at the 5′ end, the nucleus was stained with DAPI. Scale bar represented 25 μm Wu et al. BMC Molecular Biol (2019) 20:12 Page 3 of 11 the characteristics of Sf-15, we used the antibody against (named Sf-15) were constructed respectively. De novo Fibrillarin, one of the most important core protein of transcriptome sequencing was used to detect the dif- C/D box snoRNPs to pull down RNAs. Results showed ferential expression of protein coding genes. A total of that Sf-15 can be fshed out by Fibrillarin (Fig. 1c), which 59,297,730, 59,614,986 and 57,045,606 raw reads were suggested that Sf-15 might be a real snoRNA. obtained from the three libraries of Sf9, ASO and Sf-15, Next, the cellular location of Sf-15 was detected in Sf9 respectively. After removing the low-quality reads and cells. Results showed that Sf-15 was highly existed in adaptor sequences, a total of 77,578, 82,715, and 73,398 the nucleus of Sf9 cells (Fig. 1d, e). Moreover, Immuno- contigs were obtained from the three libraries, respec- fuorescence in situ hybridization (FISH) of Sf-15 with tively (Table 1). Finally, we got 46,529, 47,231 and 43,032 Cy3-labeled antisense probes confrmed that Sf-15 was unigenes from Sf9, ASO and Sf-15, respectively, with an dominantly in nucleus of Sf9 cells (Fig. 1f). average length of 750 bp. A total of 9291 (34.81%) unigenes were classifed into Repression of Sf‑15 blocks proliferation and promotes 3 main GO categories (biological process, cellular com- apoptosis and death of Sf9 cells ponent, and molecular function) and 57 subcategories snoRNAs are located in the nucleoli, the traditional (Fig. 3a, Additional fle 1: Table S1). Te large part of methods of RNA interference (RNAi) such as dsRNAs unigenes were assigned to cellular process, with a per- or siRNAs can hardly work, but RNase H1-dependent centage of 14.98%, then followed by single-organism pro- antisense oligonucleotides (ASOs) can be easily trans- cess (11.51%) and metabolic process (11.44%). A total of ported into the nucleus [35–38]. So here the modifed 17,700 COG functional annotations were obtained and ASO of Sf-15 was used to repress the expression of Sf-15 classifed into 25 functional categories (Fig. 3b). in Sf9 cells. Results showed that the expression of Sf-15 Expression analysis showed that 355 unigenes were sig- was inhibited by nearly 33% after 24 h transfection, and nifcantly upregulated and 1099 were signifcantly down- reached to 75% after 72 h transfection of ASO (Fig.