Structural analyses of NEAT1 lncRNAs suggest long-range RNA interactions that may contribute to paraspeckle architecture

Abstract

Paraspeckles are nuclear bodies that regulate multiple aspects of gene expression. The long non-coding RNA (lncRNA) NEAT1 is essential for paraspeckle formation. NEAT1 has a highly ordered spatial organization within the paraspeckle, such that its 5′ and 3′ ends localize on the periphery of paraspeckle, while central sequences of NEAT1 are found within the paraspeckle core. As such, the structure of NEAT1 RNA may be important as a scaffold for the paraspeckle. In this study, we used SHAPE probing and computational analyses to investigate the secondary structure of human and mouse NEAT1. We propose a secondary structural model of the shorter (3,735 nt) isoform hNEAT1_S, in which the RNA folds into four separate domains. The secondary structures of mouse and human NEAT1 are largely different, with the exception of several short regions that have high structural similarity. Long-range base-pairing interactions between the 5′ and 3′ ends of the long isoform NEAT1 (NEAT1_L) were predicted computationally and verified using an in vitro RNA–RNA interaction assay. These results suggest that the conserved role of NEAT1 as a paraspeckle scaffold does not require extensively conserved RNA secondary structure and that long-range interactions among NEAT1 transcripts may have an important architectural function in paraspeckle formation.

Membraneless nuclear organelles and the search for phases within phases

“…It is unclear how the localization of BRG1 in these “patches” correlates with its known ability to assembly paraspeckles (Kawaguchi et al, ). Both the electron microscopy and SRM data show that NEAT1 folds in half, with the 5′ and 3′ ends positioned in the outer shell, and the middle of the RNA in the inner core, which correlates with the self‐interaction data summarized above (Lin et al, ; Souquere et al, ; West et al, ). This correlates well with recent research that indicates the NEAT1 lncRNA middle domain is essential for NONO and SFPQ recruitment, and phase separation (Yamazaki et al, ).…”

“…The NEAT1 lncRNA gene product is multivalent and interacts directly with various paraspeckle proteins, which mediates paraspeckle nucleation and supports the hypothesis that this nuclear-retained RNA is essential for their formation (Clemson et al, 2009;Sunwoo et al, 2009). However, a recent study has suggested that long-range contacts between the 5 0 and 3 0 ends of NEAT1 RNA also contribute to paraspeckle structure (Lin, Schmidt, Bruchez, & McManus, 2018). Many paraspeckle proteins contain prion-like IDR domains, contributing to the ability to form large, phase-separated condensates in the nucleus and hydrogels in vitro (Hennig et al, 2015;Schwartz et al, 2013).…”

The how and why of lncRNA function: An innate immune perspective

“…As stated above, ONT and PacBio can perform long read cDNA sequencing [260] and even more exciting, both technologies can perform direct long read RNA sequencing [261,262]. The beauty and simplicity that ONT and PacBio offers is the ability to sequence the full expressed isoform (cDNA and direct RNA), without worries of misidentifying a complex splicing pattern, RNA cleavage events and also not relying on transcript annotation files will lead to the identification of novel isoforms which are problems faced using short-read Illumina sequencing.…”

Long non-coding RNA expression levels modulate cell-type specific splicing patterns by altering their interaction landscape with RNA-binding proteins

“…Investigations of lncRNA secondary structure and tertiary structure have revealed methods of epigenetic and post-transcriptomic regulation [38,46] and may further implicate lncRNA's influence over alternative splicing. Thereby, we propose that lncRNAs like RP5-1148A21.3 may interact intramolecularly and alter post-transcriptional gene regulatory networks.…”

“…Additionally, subnuclear bodies have shown promise in understanding lncRNA's potential in creating other cellular machinery [37], and lncRNAs which bind to many RBPs could be recruiting those proteins to form a subnuclear body. Groups invested in mechanisms of post-transcriptional regulation may begin to examine RBP binding data, and multiple sequence alignment of lncRNAs in order to understand intramolecular interactions in subnuclear bodies and secondary lncRNA structure [38,39]. Hence, further attempts to understand non-coding RNA structure, should account for interactions of lncRNAs and RBPs inside of the nucleus and in the cytoplasm in order to reveal other means of lncRNAs' involvement in splicing.…”