Structural Analyses of NEAT1 Lncrnas Suggest Long-Range RNA Interactions That May Contribute to Paraspeckle Architecture Yizhu Lin1, Brigitte F
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3742–3752 Nucleic Acids Research, 2018, Vol. 46, No. 7 Published online 31 January 2018 doi: 10.1093/nar/gky046 Structural analyses of NEAT1 lncRNAs suggest long-range RNA interactions that may contribute to paraspeckle architecture Yizhu Lin1, Brigitte F. Schmidt2, Marcel P. Bruchez1,2,3 and C. Joel McManus1,* 1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA, 2Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA and 3Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA Received April 03, 2017; Revised December 20, 2017; Editorial Decision January 04, 2018; Accepted January 29, 2018 ABSTRACT tory functions through diverse mechanisms including chro- matin binding (Xist) (2), regulating gene transcription in cis Paraspeckles are nuclear bodies that regulate multi- (ANRIL) (3), and scaffolding of nuclear bodies (NEAT1). ple aspects of gene expression. The long non-coding Intriguingly, although many lncRNA have important con- RNA (lncRNA) NEAT1 is essential for paraspeckle served functions, they usually have relatively low sequence formation. NEAT1 has a highly ordered spatial or- conservation (1). This is counterintuitive, as sequence con- ganization within the paraspeckle, such that its 5 servation is often assumed to be required for genes with and 3 ends localize on the periphery of paraspeckle, important functions (4). One possible explanation is that while central sequences of NEAT1 are found within lncRNA preserve higher order conservation, such as con- the paraspeckle core. As such, the structure of servation of secondary structure (base pairing interactions) NEAT1 RNA may be important as a scaffold for the or tertiary structure (three dimensional shape of folded paraspeckle. In this study, we used SHAPE probing RNA). Large RNAs fold into secondary structures, which then and computational analyses to investigate the sec- influence their 3D tertiary structures. Resolving the sec- ondary structure of human and mouse NEAT1. We ondary structures of lncRNAs in vivo is a difficult task propose a secondary structural model of the shorter due to their large size and low abundance in cells. High- (3,735 nt) isoform hNEAT1 S, in which the RNA folds throughput in vivo structure probing using reverse tran- into four separate domains. The secondary struc- scription truncation (-seq) methods requires extreme se- tures of mouse and human NEAT1 are largely dif- quence depth for low abundance lncRNAs. Till now, there ferent, with the exception of several short regions is only one human lncRNA, Xist, whose structure has been that have high structural similarity. Long-range base- probed in vivo (5). Furthermore, lncRNAs are expressed in pairing interactions between the 5 and 3 ends of the alternative isoforms and bound by a variety of RNA bind- long isoform NEAT1 (NEAT1 L) were predicted com- ing proteins in vivo, both of which can obscure interpre- putationally and verified using an in vitro RNA–RNA tation of chemical modification patterns. In vitro structure probing interrogates an RNA’s inherent folding potential interaction assay. These results suggest that the con- without interference by bound proteins or alternative tran- served role of NEAT1 as a paraspeckle scaffold does script isoforms. Although this simplifies the task, the large not require extensively conserved RNA secondary size of lncRNA still poses a significant challenge, and only structure and that long-range interactions among a few lncRNA structures have been experimentally charac- NEAT1 transcripts may have an important architec- terized in vitro (6) (HOTAIR (7), Xist (8,9) and ncSRA (10) tural function in paraspeckle formation. RepA (11) and lincRNAp21 (12)). NEAT1 is an especially interesting lncRNA for structural study. It is a key structural component of paraspeckles and INTRODUCTION is essential for paraspeckle formation. Paraspeckles are nu- Long non-coding RNAs (lncRNAs) are defined as non- clear bodies located in the nucleus interchromatin space. protein coding RNAs that are longer than 200 nucleotides. Though paraspeckle functions and regulatory mechanisms In the human genome, more than thirteen thousand lncR- are not completely understood, recent studies showed they NAs have been annotated (1), making up a large propor- are involved in multiple gene regulatory processes, such tion of human genes. lncRNAs are involved in gene regula- as mRNA retention, mRNA cleavage, A-to-I editing (13) *To whom correspondence should be addressed. Tel: +1 412 268 9407; Email: [email protected] C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2018, Vol. 46, No. 7 3743 and protein sequestration (14). These regulatory functions and identify a strong propensity for long-range intramolec- are responsible for several cellular responses and shown to ular base-pairing that may contribute to scaffolding the be associated with the pathology of multiple cancers and paraspeckle. neurodegenerative diseases (15–17). Deletion of NEAT1 in mice disrupts development of female reproductive tissues, MATERIALS AND METHODS underscoring the biological importance of this lncRNA (18,19). In vitro transcription NEAT1 has two isoforms that share the same transcrip- hNEAT1 S and mNEAT1 S plasmids were generously pro- tion start site, but have different termination sites. In hu- vided by Dr Gerard´ Pierron (27) and Dr Lingling Chen (28), mans, the short isoform NEAT1 S is 3735 nt long with respectively. PCR primers were designed for both full length a polyA tail. The long isoform, which is essential for NEAT1 RNA and short segments, and the SP6 promoter paraspeckle formation, is 22 741 nt in length and has a sequence was included in the forward primers. The DNA non-polyadenylated 3 end produced by RNase P cleavage template for in vitro transcription was amplified from the (20,21). The expression level of NEAT1 Sisestimatedto plasmids using Phusion high-fidelity polymerase and puri- be at least five-fold higher than NEAT1 L, and even higher fied by agarose gel extraction. The RNA was in vitro tran- in many tissues and cell types (22,23). Though less abun- scribed using Promega RiboMAX large scale RNA pro- dant, NEAT1 L is considered to be the key isoform for duction systems (SP6), as described in the manufacturer’s paraspeckle formation. Targeted knock down of NEAT1 L instructions. Briefly, 200–500 ng cDNA template, 4 l5X leads to loss of paraspeckles, while de novo paraspeckle for- SP6 buffer, 4 l25mMrNTPsand2l SP6 enzyme mix mation can be rescued by transient expression of NEAT1 L were mixed in a 20 l reaction and incubated at 37◦C for 3.5 (20,24). Intriguingly, NEAT1 S can be found outside of the hours. 0.5 l RQ1 RNase-Free DNase (1u/l) were added paraspeckle in tissue culture cells, suggesting it may have in- to each reaction and incubated at 37◦C for 15 min to de- dependent biological functions (25). The two isoform gene stroy DNA template. 0.5 l proteinase K (20 mg/ml) was structure and the function of NEAT1 in paraspeckle for- then added to reaction and incubated at 37◦C for 1 h to de- mation were observed in both humans and mice. However, stroy SP6 transcriptase and RQ1 DNase. the sequence of NEAT1 is not well conserved between hu- man and mouse. This suggests higher-order conservation of NEAT1 RNAs, such as secondary structural conservation Non-denaturing purification of RNA or conserved RNA-protein interactions. A non-denaturing purification was adapted from So- Interestingly, evidence has emerged indicating that the marowthu et al. (7) to maintain the co-transcriptionally specific structural conformation of NEAT1 might be folded structure for SHAPE probing experiments. Briefly, important for paraspeckle architecture. EM-ISH (elec- after proteinase K treatment, the RNA was diluted with 200 tron microscopy-in situ hybridization) studies using DNA × l1 SHAPE buffer (111 mM NaCl, 111 mM HEPES, probes to the 5 and 3 ends of NEAT1 LRNAshowedthat 6.67 mM MgCl2), transferred to Amicon Ultra 100K col- NEAT1 L has a highly ordered spatial organization within umn and centrifuged at 14 000 g for 10 min to con- the paraspeckle (15). The 5 and 3 ends of NEAT1 Lwere centrate the RNA sample to approximately 30 l. This localized to the paraspeckle periphery, while the central re- dilution/concentration step was repeated for a total of two gion of NEAT1 L was found within the paraspeckle core. rounds. The purified RNA was then collected by centrifug- Since the 5 end of NEAT1 L is identical to NEAT1 S, the ing the column upside down 2 min at 1000g. The RNAs short isoform NEAT1 S should also localize to the periph- were verified on a TapeStation. The RNAs were kept on ice ery of paraspeckle. Based on these observations, an ultra- and were immediately used for SHAPE probing structural paraspeckle model was proposed with two salient features. First, NEAT1 L folds end-to-end. Secondly, mul- 1M7 synthesis procedure tiple folded NEAT1 L and NEAT1 S molecules are regu- larly organized in the cross sections of paraspeckle, forming We synthesized 1M7 using a novel procedure. In a circular skeleton. However, the actual secondary structure brief, 2-amino-4-nitrobenzoic acid was converted to 2- of NEAT1 has not yet been characterized. The nature of ((ethoxycarbonyl)amino)-4-nitrobenzoic acid through the the spatial organization of NEAT1 and its contribution to addition of ethyl chloroformate by reflux for 1 h. This paraspeckle architecture is yet to be understood. product was converted to 7-nitro-1H-benzo[d][1,3]oxazine- Here, we combined high throughput RNA structure 2,4-dione by heating at 65◦C in the presence of thionylchlo- probing (Mod-seq) (26) with computational analyses to in- ride for 30 min, cooled to room temperature and washed vestigate the structural features of NEAT1.