Paraspeckles: nuclear bodies based on a NEAT1 structure

RNAs are essential catalysts and regulators at every step of gene expression. Many new classes of ncRNAs have been described in the past years. Especially long non-coding RNAs (lncRNAs) display a wide variety of functions, ranging from scaffolding to guiding and signalling. LncRNAs are implicated in different disease pathologies in higher eukaryotes triggered by single nucleotide polymorphisms which presumably affect lncRNA conformation and/or their interactions with protein partners. Despite their biomedical significance, the structure of lncRNAs and its implication for function remains largely unknown. NEAT1 is the scaffolding lncRNA of nuclear bodies called paraspeckles, which have been involved in the cellular stress response and differentiation. Paraspeckles are dynamic structures that assemble newly after every cell cycle and increase in size and number when responding to stress. They contain a special RNA-binding protein family, the Drosophila behavior/human splicing (DBHS) proteins, which are important for paraspeckle biogenesis and maintenance. Interestingly, biogenesis of paraspeckles occurs co-transcriptionally at the NEAT1 site and requires two of the DBHS proteins. The interactions of RNA-RNA as well as RNA-proteins in paraspeckles are poorly understood and solely rely on immunoprecipitation experiments. To understand the molecular architecture of paraspeckles, I will use chemical probing to map the structure of the short NEAT1 transcript that has been implicated in paraspeckle nucleation. This data will provide the first in vivo structure of a lncRNA. Furthermore, I aim at detecting possible conformational changes within the short NEAT1 RNA induced by the core paraspeckle protein NONO. These changes are the result of either direct RNA-protein contacts or of novel intramolecular interactions of NEAT1 that can only form in the presence of NONO. Moreover, I will determine which of the core paraspeckle proteins bind NEAT1 directly and where these interactions occur by using high-throughput sequencing together with UV-crosslinking and immunoprecipitation (HITS-CLIP). Together with the in vivo fold of the short NEAT1, this data will enable me to shed light on the molecular architecture of this ribonucleoprotein complex. Overall, my data will give first insights about the structure of a scaffolding lncRNA and how interactions with its binding partners shape the architecture of a nuclear body.

Scientific Background: Ribonucleic acids (RNAs) are essential catalysts and regulators at every step of gene expression. Many new classes of noncoding RNAs (ncRNAs) have been described in the past years. Especially long non-coding RNAs (lncRNAs) display a wide variety of functions, ranging from scaffolding to guiding and signaling. LncRNAs are implicated in different disease pathologies in higher eukaryotes triggered by single nucleotide polymorphisms which presumably affect lncRNA structure and/or their interactions with protein partners. Despite their biomedical significance, the structure of lncRNAs and its implication for function remain largely unknown. Project Focus: NEAT1 is the scaffolding lncRNA of nuclear bodies called paraspeckles, which have been involved in the cellular stress response and differentiation. Paraspeckles are dynamic structures that assemble newly after every cell cycle and increase in size and number when responding to stress. They contain a special RNA-binding protein family, the Drosophila behavior/human splicing (DBHS) proteins, which are important for paraspeckle biogenesis and maintenance. Project aims: The study aimed at understanding how the short NEAT1 RNA folds in vivo and interacts with paraspeckle core proteins to form paraspeckles. Especially, the interaction with core paraspeckle protein NONO was pivotal since it is essential for the biogenesis and maintenance of paraspeckles. As there are two NEAT1 transcript variants described, a long and a short form, it was of utmost interest to depict the individual function and contribution of the short NEAT1 RNA in the architecture of paraspeckles and their biological roles in the cell. Project results: The initial hypothesis that the short NEAT1 RNA is important for paraspeckle biogenesis or maintenance proved to be wrong. The work of Li et al. (2017) demonstrated that cells only expressing the short NEAT1 RNA showed no presence of paraspeckles. The short NEAT1 RNA localized in numerous non-paraspeckle foci termed microspeckles, but these did not contain the core paraspeckle protein NONO. Consequently, the short NEAT1 RNA is not involved in paraspeckle construction. The mapping of the short NEAT1 RNA proved technically unattainable in vivo. While working on the in vitro folding of the short NEAT1 RNA, Lin et al. (2018) published beforehand. Not only did their in vitro structural mapping show a complete folding back of both ends of the short NEAT1 RNA on themselves, but also the formation of four domains within the short NEAT1 RNA were formed. Their data confirmed our in silico folding of the short NEAT1 RNA using the RNAfold software (ViennaRNA Package).