DOCSLIB.ORG

Small RNA Sequencing Reveals Distinct Nuclear Micrornas in Pig Granulosa Cells During Ovarian Follicle Growth Derek Toms1* , Bo Pan2, Yinshan Bai2,3 and Julang Li2

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Small RNA Sequencing Reveals Distinct Nuclear Micrornas in Pig Granulosa Cells During Ovarian Follicle Growth Derek Toms1* , Bo Pan2, Yinshan Bai2,3 and Julang Li2 Toms et al. Journal of Ovarian Research (2021) 14:54 https://doi.org/10.1186/s13048-021-00802-3 RESEARCH Open Access Small RNA sequencing reveals distinct nuclear microRNAs in pig granulosa cells during ovarian follicle growth Derek Toms1* , Bo Pan2, Yinshan Bai2,3 and Julang Li2 Abstract Nuclear small RNAs have emerged as an important subset of non-coding RNA species that are capable of regulating gene expression. A type of small RNA, microRNA (miRNA) have been shown to regulate development of the ovarian follicle via canonical targeting and translational repression. Little has been done to study these molecules at a subcellular level. Using cell fractionation and high throughput sequencing, we surveyed cytoplasmic and nuclear small RNA found in the granulosa cells of the pig ovarian antral preovulatory follicle. Bioinformatics analysis revealed a diverse network of small RNA that differ in their subcellular distribution and implied function. We identified predicted genomic DNA binding sites for nucleus-enriched miRNAs that may potentially be involved in transcriptional regulation. The small nucleolar RNA (snoRNA) SNORA73, known to be involved in steroid synthesis, was also found to be highly enriched in the cytoplasm, suggesting a role for snoRNA species in ovarian function. Taken together, these data provide an important resource to study the small RNAome in ovarian follicles and how they may impact fertility. Keywords: Microrna, smallRNA, snoRNA, Granulosa cells, Pig, Ovarian, Ovary, Follicle, Reproductive biology, Next generation sequencing, RNA-seq, Cellular fractionation, Nucleus, Cytoplasm Introduction species are transcribed from chromosomal DNA, and Increasing ovarian follicle size has long been an import- most are processed in both the nucleus and cytoplasm. ant indicator of oocyte quality, with oocytes obtained Accordingly, a complex system of transport exists to from large follicles showing consistently higher in vitro dynamically localize small RNA within the cell. For ex- and in vivo developmental potential than their counter- ample, after processing into mature miRNA by Dicer in parts from small follicles [3, 51]. In addition to increased the cytoplasm, strands loaded onto Ago2 may be subse- expression of key growth factors like GDF9 and BMP4, quently imported back into the nucleus via Importin 8 we and others have shown differences in microRNA [39]. The availability of complex secondary structures (miRNA) expression in the somatic cells of these follicles between RNA and DNA species permit these RNA to [44–46, 54, 55, 57]. Recent investigations have looked at function in a myriad of ways. In the nucleus, miRNA the distinct populations of small RNA that perform key can form triple helices with chromosomal DNA, to regu- biological functions in the cytoplasmic and nuclear com- late gene expression [48]. Even degraded fragments of partments of cells [19, 41, 47, 60]. All of these RNA miscoded messenger RNA have been shown to partici- pate in the assembly of such scaffolds [9]. Although * Correspondence: [email protected] small nucleolar RNAs (snoRNAs) are principally in- 1Department of Comparative Biology and Experimental Medicine, Faculty of volved with RNA modification in the nucleus, they are Veterinary Medicine, University of Calgary, Calgary, AB, Canada shuttled to the cytoplasm during periods of stress [20, Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Toms et al. Journal of Ovarian Research (2021) 14:54 Page 2 of 12 31]. Despite evidence that such RNA species have func- lower in the nuclear fraction compared to cytoplasmic tional roles in both the nuclear and cytoplasmic com- fractions. partments of the cell, little systematic analysis has been done to study them at a population level. Here, we MicroRNAs have distinct subcellular localization patterns present a survey of small RNA species present in gilt After mapping reads to annotated swine miRNA, we granulosa cells obtained from small and large preovula- looked at the relationship between samples using princi- tory follicles, herein referred to as SGCs and LGCs. pal component analysis and unsupervised clustering. While we found a few significant differences between The first principle component comprised 54% of the SGCs and LGCs, we did reveal a diverse network of variance and separated nuclear samples from cytoplas- small RNA that showed distinct subcellular localization. mic samples. The second principal component that com- This small RNAome of ovarian granulosa cells will pro- prised 11% of the variance in the data was related to vide an important resource for studying their subcellular batch effect, although interestingly this primarily affected function during follicle growth. the variance of nuclear samples. This batch effect was corrected for in subsequent statistical analysis. Expres- Results sion of miRNA from distinct subcellular components Validation of subcellular fractionation clustered together without any obvious differences be- Following cell fractionation, intact nuclei were visualized tween miRNA from SGC and LGC (Fig. 3a). We next by phase-contrast and fluorescence microscopy, after examined expression of miRNA using a generalized lin- chromosome staining with Hoescht 33342 (Fig. 1a). Be- ear model (see Methods) comparing all four groups fore proceeding with deep sequencing of small RNAs, (SGC nuclei, LGC nuclei, SGC cytoplasm, and LGC we confirmed the quality and purity of the nuclear and cytoplasm). While most miRNA that showed differences cytoplasmic fractions. The RNA integrity number (RIN) in expression between the nucleus and cytoplasm were for each sample was determined by capillary electro- common between SGC and LGC, several miRNA dif- phoresis: LGC cytosol, 8.62 ± 0.40; LGC nucleus, 6.92 ± fered in their subcellular localization between granulosa 0.17; SGC cytosol, 9.02 ± 0.22; SGC nucleus, 6.78 ± 0.46. cells from the two stages of follicular development. Fur- Western blotting revealed the purity of each fraction by ther examination of nuclear miRNA revealed seven dif- the exclusion of glyceraldehyde 3-phosphate dehydro- ferentially expressed between SGC and LGC (Fig. 3b). genase from the nucleus and lamin B from the cytosol We then ranked miRNA based on their fold change (Fig. 1b). RT-qPCR analysis of the nuclear spliceosomal between the nucleus and cytoplasm (Supplemental Ta- RNA U6 revealed 1000-fold higher expression in the nu- bles 1 and 2). SGC showed more significantly different cleus, as expected (Fig. 1c), confirming clear separation miRNAs between these subcellular compartments than of the nucleus and cytosol fractions. LGC (101 versus 83, respectively). To confirm our sequencing results, we analyzed ex- Overview of small RNA sequencing data pression of 12 miRNA by digital droplet RT-PCR in our Four nuclear and four cytoplasmic RNA pools were se- fractionated samples. All analyzed miRNA showed iden- quenced from both LGCs and SGCs. After trimming tical enrichment, either cytoplasmic or nuclear, and a adapter sequences, reads with a sequence length less significant correlation of expression ratios (R = 0.50, P = than 18 nt were discarded. Remaining sequencing reads 0.013) between the two technologies (Fig. 4). had lengths between 18 and 50 nt (Fig. 2a). The majority of reads (97%) were between 22 and 25 nt in length. Some nuclear miRNAs are predicted to target promoter Twenty percent of these reads could not be mapped to regions the pig genome and 7% mapped to coding regions. With specific miRNAs observed to be enriched in the We mapped the remaining reads to S. scrofa small nucleus of granulosa cells and previous work demon- RNA databases and looked broadly at RNA species strating that small RNA in the nucleus can regulate tran- breakdown in nuclear and cytoplasmic fractions (Fig. 2b). scription, we examined the potential targeting of these MicroRNA, both mature sequences and hairpin precur- nuclear miRNAs to genomic promoter regions. Because sors, made up the majority of the mapped reads in both of a dearth of annotation for pig promoter regions, we cell fractions (55% of the nuclear reads; 61% of cytoplas- used the fact that mature miRNAs are highly homolo- mic reads) followed by 30.4% of nuclear reads mapping gous [13] and looked first at binding sites in human to snoRNA, while 16.3% of cytoplasmic reads remained promoter regions that were then mapped to homologous uncharacterized as “miscellaneous RNA”. As expected, regions in the pig. Our analysis revealed 417 potential considerably fewer cytoplasmic reads mapped to binding sites at the promoter regions across the human snoRNA, while reads mapping to ribozyme, transfer genome, of which 55 had identical matches to homolo- RNA and mitochondrial RNA were five- to ten-fold gous regions in the pig genome (Supplemental Table 3 Toms et al. Journal of Ovarian Research (2021) 14:54 Page 3 of 12 Fig. 1 Isolation of nuclear and cytoplasmic fractions. a Isolated nuclei were visualized at 400X magnification to confirm purity, left-to-right: phase contrast, Hoescht 33342, and trypan blue.
Load more

Recommended publications
  • Human 28S Rrna 5' Terminal Derived Small RNA Inhibits Ribosomal Protein Mrna Levels Shuai Li 1* 1. Department of Breast Cancer
    bioRxiv preprint doi: https://doi.org/10.1101/618520; this version posted April 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Human 28s rRNA 5’ terminal derived small RNA inhibits ribosomal protein mRNA levels Shuai Li 1* 1. Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China. * To whom correspondence should be addressed. Tel.: +86 22 23340123; Fax: +86 22 23340123; Email: [email protected] & [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/618520; this version posted April 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Recent small RNA (sRNA) high-throughput sequencing studies reveal ribosomal RNAs (rRNAs) as major resources of sRNA. By reanalyzing sRNA sequencing datasets from Gene Expression Omnibus (GEO), we identify 28s rRNA 5’ terminal derived sRNA (named 28s5-rtsRNA) as the most abundant rRNA-derived sRNAs. These 28s5-rtsRNAs show a length dynamics with identical 5’ end and different 3’ end. Through exploring sRNA sequencing datasets of different human tissues, 28s5-rtsRNA is found to be highly expressed in bladder, macrophage and skin. We also show 28s5-rtsRNA is independent of microRNA biogenesis pathway and not associated with Argonaut proteins. Overexpression of 28s5-rtsRNA could alter the 28s/18s rRNA ratio and decrease multiple ribosomal protein mRNA levels.
    [Show full text]
  • Methods in and Applications of the Sequencing of Short Non-Coding Rnas" (2013)
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Methods in and Applications of the Sequencing of Short Non- Coding RNAs Paul Ryvkin University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ryvkin, Paul, "Methods in and Applications of the Sequencing of Short Non-Coding RNAs" (2013). Publicly Accessible Penn Dissertations. 922. https://repository.upenn.edu/edissertations/922 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/922 For more information, please contact [email protected]. Methods in and Applications of the Sequencing of Short Non-Coding RNAs Abstract Short non-coding RNAs are important for all domains of life. With the advent of modern molecular biology their applicability to medicine has become apparent in settings ranging from diagonistic biomarkers to therapeutics and fields angingr from oncology to neurology. In addition, a critical, recent technological development is high-throughput sequencing of nucleic acids. The convergence of modern biotechnology with developments in RNA biology presents opportunities in both basic research and medical settings. Here I present two novel methods for leveraging high-throughput sequencing in the study of short non- coding RNAs, as well as a study in which they are applied to Alzheimer's Disease (AD). The computational methods presented here include High-throughput Annotation of Modified Ribonucleotides (HAMR), which enables researchers to detect post-transcriptional covalent modifications ot RNAs in a high-throughput manner. In addition, I describe Classification of RNAs by Analysis of Length (CoRAL), a computational method that allows researchers to characterize the pathways responsible for short non-coding RNA biogenesis.
    [Show full text]
  • Small Non-Coding Rnaome of Ageing Chondrocytes
    International Journal of Molecular Sciences Article Small Non-Coding RNAome of Ageing Chondrocytes Panagiotis Balaskas 1,* , Jonathan A. Green 2, Tariq M. Haqqi 2, Philip Dyer 1, Yalda A. Kharaz 1, Yongxiang Fang 3 , Xuan Liu 3, Tim J.M. Welting 4 and Mandy J. Peffers 1,* 1 Institute of Life Course and Medical Sciences, William Henry Duncan Building, 6 West Derby Street, Liverpool L7 8TX, UK; [email protected] (P.D.); [email protected] (Y.A.K.) 2 Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, USA; [email protected] (J.A.G.); [email protected] (T.M.H.) 3 Centre for Genomic Research, Institute of Integrative Biology, Biosciences Building, Crown Street, University of Liverpool, Liverpool L69 7ZB, UK; [email protected] (Y.F.); [email protected] (X.L.) 4 Department of Orthopaedic Surgery, Maastricht University Medical Centre, 6202 AZ Maastricht, The Netherlands; [email protected] * Correspondence: [email protected] (P.B.); peff[email protected] (M.J.P.); Tel.: +44-0787-269-2102 (M.J.P.) Received: 20 July 2020; Accepted: 4 August 2020; Published: 7 August 2020 Abstract: Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5.
    [Show full text]
  • Reproducibility of High-Throughput Mrna and Small RNA Sequencing Across Laboratories
    ARTICLES Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories Peter A C ’t Hoen1,2, Marc R Friedländer3–6,15, Jonas Almlöf7,15, Michael Sammeth3–5,8,14, Irina Pulyakhina1, Seyed Yahya Anvar1,9, Jeroen F J Laros1,2,9, Henk P J Buermans1,9, Olof Karlberg7, Mathias Brännvall7, The GEUVADIS Consortium10, Johan T den Dunnen1,2,9, Gert-Jan B van Ommen1, Ivo G Gut8, Roderic Guigó3–5, Xavier Estivill3–6, Ann-Christine Syvänen7, Emmanouil T Dermitzakis11–13 & Tuuli Lappalainen11–13 RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and guidelines for assessing technical biases in RNA-seq data. RNA sequencing (RNA-seq) has transformed the field of transcrip- a set of essential quality measures for RNA-seq experiments and dis- tomics1–4.
    [Show full text]
  • Improved TGIRT-Seq Methods for Comprehensive Transcriptome Profiling with Decreased Adapter Dimer Formation and Bias Correction
    bioRxiv preprint doi: https://doi.org/10.1101/474031; this version posted November 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction 1,2,3 1,2,3 1,2,3 1,2 Hengyi Xu, Jun Yao, Douglas C. Wu and Alan M. Lambowitz 1Institute for Cellular and Molecular Biology University of Texas at Austin Austin Texas, 78712, USA 2Department of Molecular Biosciences, University of Texas at Austin, Austin Texas, 78712, USA Running Head: Improved TGIRT-seq methods 3These authors contributed equally to this work. Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/474031; this version posted November 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA- seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5’- and 3’-end biases.
    [Show full text]
  • Genapsys™ Sequencing Platform: Small RNA Sequencing
    TECHNICAL NOTE GenapSys™ Sequencing Platform: Small RNA Sequencing 2 • Interrogate thousands of small RNAs in parallel R =0.990 with greater sensitivity and dynamic range vs 105 orthogonal methods • Sample batching to enable cost-effective 4 small RNA sequencing 10 • Utilize an affordable, personal NGS platform that offers a low price per run and low price per sample 103 • Leverage a range of library prep kits as part of a simple, flexible workflow solution 102 Introduction Illumina (Read Count) 101 Small RNAs are important non-coding regulators in diverse biological settings. MicroRNAs (miRNAs) that are the 101 102 103 104 105 most widely studied small RNAs regulate protein-coding expression post-transcriptionally. miRNA expression profiles GenapSys (Read Count) in tissues or cell populations are highly informative to reveal Fig. 1 Strong correlation between GenapSys and Illumina sequencing cellular states, such as in human cancers, and to identify of a small RNA library. A library was generated with the Universal Human cellular mechanisms. Small RNAs, including miRNAs, miRNA Reference RNA (Agilent) using the QiaSeq miRNA library prep kit. The final PCR step was performed with a GenapSys-customized PCR are single-stranded non-coding RNAs between 17-34 primer instead of the library PCR primer from the kit. nucleotides in length and are involved in the regulation of numerous cellular processes. The GenapSys™ Sequencing species altogether. By comparison, NGS offers an ability to Platform offers the most affordable platform for small count individual transcripts enabling a large dynamic range. RNA sequencing. Researchers can choose the level of sensitivity that is needed for their experiment allowing for optimization for sensitivity Next-Generation Sequencing (NGS) offers a digital readout or the number of samples that can be processed per run.
    [Show full text]
  • RNA-Sequencing Analyses of Small Bacterial Rnas and Their Emergence As Virulence Factors in Host-Pathogen Interactions
    International Journal of Molecular Sciences Review RNA-Sequencing Analyses of Small Bacterial RNAs and their Emergence as Virulence Factors in Host-Pathogen Interactions Idrissa Diallo and Patrick Provost * CHUQ Research Center/CHUL, Department of Microbiology-Infectious Disease and Immunity, Faculty of Medicine, Université Laval, Quebec, QC G1V 0A6, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-418-525-4444 (ext. 48842) Received: 1 February 2020; Accepted: 25 February 2020; Published: 27 February 2020 Abstract: Proteins have long been considered to be the most prominent factors regulating so-called invasive genes involved in host-pathogen interactions. The possible role of small non-coding RNAs (sRNAs), either intracellular, secreted or packaged in outer membrane vesicles (OMVs), remained unclear until recently. The advent of high-throughput RNA-sequencing (RNA-seq) techniques has accelerated sRNA discovery. RNA-seq radically changed the paradigm on bacterial virulence and pathogenicity to the point that sRNAs are emerging as an important, distinct class of virulence factors in both gram-positive and gram-negative bacteria. The potential of OMVs, as protectors and carriers of these functional, gene regulatory sRNAs between cells, has also provided an additional layer of complexity to the dynamic host-pathogen relationship. Using a non-exhaustive approach and through examples, this review aims to discuss the involvement of sRNAs, either free or loaded in OMVs, in the mechanisms of virulence and pathogenicity during bacterial infection. We provide a brief overview of sRNA origin and importance and describe the classical and more recent methods of identification that have enabled their discovery, with an emphasis on the theoretical lower limit of RNA sizes considered for RNA sequencing and bioinformatics analyses.
    [Show full text]
  • Automated Analysis of Small RNA Datasets with RAPID
    bioRxiv preprint doi: https://doi.org/10.1101/303750; this version posted February 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Automated analysis of small RNA datasets with RAPID Sivarajan Karunanithi1;2;4, Martin Simon3;6, Marcel H. Schulz1;5 February 20, 2019 1 Cluster of Excellence for Multimodal Computing and Interaction, Saarland University and Department for Computational Biology & Applied Algorithmics, Max Planck Institute for In- formatics, Saarland Informatics Campus, Saarbr¨ucken, Germany. 2 Graduate School of Computer Science, Saarland University, Saarland Informatics Campus, Saarbr¨ucken, Germany. 3 Molecular Cell Dynamics Saarland University, Centre for Human and Molecular Biology, Saar- land University, Saarbr¨ucken, Germany. 4 Institute for Cardiovascular Regeneration, Goethe-University Hospital, Theodor Stern Kai 7, Frankfurt, Germany. 5 German Centre for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt, Ger- many. 6 Molecular Cell Biology and Microbiology, Wuppertal University, Gausstraße 20, Wuppertal, Germany. Abstract Summary: Understanding the role of short-interfering RNA (siRNA) in diverse biolog- ical processes is of current interest and often approached through small RNA sequencing. However, analysis of these datasets is difficult due to the complexity of biological RNA processing pathways, which differ between species. Several properties like strand specificity, length distribution, and distribution of soft-clipped bases are few parameters known to guide researchers in understanding the role of siRNAs. We present RAPID, a generic eukaryotic siRNA analysis pipeline, which captures information inherent in the datasets and automat- ically produces numerous visualizations as user-friendly HTML reports, covering multiple categories required for siRNA analysis.
    [Show full text]
  • Analysis of Small RNA Changes in Different Brassica Napus Synthetic Allopolyploids
    Analysis of small RNA changes in different Brassica napus synthetic allopolyploids Yunxiao Wei, Fei Li, Shujiang Zhang, Shifan Zhang, Hui Zhang and Rifei Sun Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China ABSTRACT Allopolyploidy is an evolutionary and mechanisticaly intriguing process involving the reconciliation of two or more sets of diverged genomes and regulatory interactions, resulting in new phenotypes. In this study, we explored the small RNA changes of eight F2 synthetic B. napus using small RNA sequencing. We found that a part of miRNAs and siRNAs were non-additively expressed in the synthesized B. napus allotetraploid. Differentially expressed miRNAs and siRNAs differed among eight F2 individuals, and the differential expression of miR159 and miR172 was consistent with that of flowering time trait. The GO enrichment analysis of differential expression miRNA target genes found that most of them were concentrated in ATP-related pathways, which might be a potential regulatory process contributing to heterosis. In addition, the number of siRNAs present in the offspring was significantly higher than that of the parent, and the number of high parents was significantly higher than the number of low parents. The results have shown that the differential expression of miRNA lays the foundation for explaining the trait separation phenomenon, and the significant increase of siRNA alleviates the shock of the newly synthesized allopolyploidy. It provides a new perspective between small RNA changes
    [Show full text]
  • Removing Bias Against Short Sequences Enables Northern Blotting to Better Complement RNA-Seq for the Study of Small Rnas
    bioRxiv preprint doi: https://doi.org/10.1101/068031; this version posted August 5, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs Yun S. Choi, Lanelle O. Edwards, Aubrey DiBello, and Antony M. Jose* Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA * To whom correspondence should be addressed. Tel: 301-405-7028; Email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/068031; this version posted August 5, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. ABSTRACT Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences, and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm C. elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq.
    [Show full text]
  • Extensive Genomic Plasticity Identified by Anti-Cancer Huaier Therapy
    Extensive genomic plasticity identied by anti- cancer Huaier therapy Manami Tanaka ( [email protected] ) Bradeion Institute of Medical Sciences, Co. Ltd. Tomoo Tanaka Bradeion Institute of Medical Sciences, Co. Ltd. Fei Teng BGI-Shenzhen Hong Lin BGI-Shenzhen Chenying Xu BGI-Shenzhen Ning Li BGI-Japan Zhu Luo BGI-Japan Ding Wei Japan Kampo NewMedicine, Co. Ltd. Zhengxin Lu QiDong Gaitianli Medicines Co. Ltd. Biological Sciences - Article Keywords: Huaier (Trametes robiniophila murr), Cancer therapy, Total RNA- and small RNA-sequencing, RNA editing events, Transcriptional control Posted Date: October 22nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-91260/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Title: Extensive genomic plasticity identified by anti-cancer Huaier therapy Manami Tanaka1)*, Tomoo Tanaka1), Fei Teng2), Hong Lin2), Chenying Xu2), Ning Li3), Zhu Luo3), Ding Wei4), and Zhengxin Lu5) 1) Bradeion Institute of Medical Sciences, Co. Ltd., Itado 344-1, Hiratsuka, Kanagawa 259-1211, Japan. 2) BGI-Shenzhen, Building NO.7, BGI Park, No.21 Hongan 3rd Street, Yantian District, Shenzhen 518083, China 3) BGI-Japan, Kobe KIMEC Center BLDG. 8F 1-5-2 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047 Japan. 4) Japan Kampo NewMedicine, Co. Ltd., 2-8-10 Kayaba-Cho, Chuo-Ku, Tokyo 103−0025, Japan. 5) QiDong Gaitianli Medicines Co. Ltd., Jiangsu Province, China. Key words: Huaier (Trametes robiniophila murr) Cancer therapy Total RNA- and small RNA-sequencing RNA editing events Transcriptional control 1 Clinical significance of anti-cancer effects of Huaier has been emphasized recently. We have proved that a broad spectrum of Huaier effects was based on the rescue of the disrupted Hippo signalling pathway, especially through the rescue of transcriptional dysregulation.
    [Show full text]
  • Distinct Mirna Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells
    International Journal of Molecular Sciences Article Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells Panayiota L. Papasavva 1,2, Nikoletta Y. Papaioannou 1,2, Petros Patsali 1,2 , Ryo Kurita 3, Yukio Nakamura 3, Maria Sitarou 4, Soteroulla Christou 5, Marina Kleanthous 1,2,† and Carsten W. Lederer 1,2,*,† 1 Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia 2371, Cyprus; [email protected] (P.L.P.); [email protected] (N.Y.P.); [email protected] (P.P.); [email protected] (M.K.) 2 Cyprus School of Molecular Medicine, Nicosia 2371, Cyprus 3 Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan; [email protected] (R.K.); [email protected] (Y.N.) 4 Thalassemia Clinic Larnaca, Larnaca General Hospital, Larnaca 6301, Cyprus; [email protected] 5 Thalassemia Clinic Nicosia, Archbishop Makarios III Hospital, Nicosia 1474, Cyprus; [email protected] * Correspondence: [email protected] † Contributed equally to this work. Abstract: MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythro- Citation: Papasavva, P.L.; poiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy Papaioannou, N.Y.; Patsali, P.; Kurita, development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively R.; Nakamura, Y.; Sitarou, M.; characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood- Christou, S.; Kleanthous, M.; Lederer, derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin C.W.
    [Show full text]