Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

Genome-wide screening of circadian and non-circadian impact of Neat1 genetic deletion.

The functions of the long non-coding RNA, Nuclear enriched abundant transcript 1 (Neat1), are poorly understood. Neat1 is required for the formation of paraspeckles, but its respective paraspeckle-dependent or independent functions are unknown. Several studies including ours reported that Neat1 is involved in the regulation of circadian rhythms. We characterized the impact of Neat1 genetic deletion in a rat pituitary cell line. The mRNAs whose circadian expression pattern or expression level is regulated by Neat1 were identified after high-throughput RNA sequencing of the circadian transcriptome of wild-type cells compared to cells in which Neat1 was deleted by CRISPR/Cas9. The numerous RNAs affected by Neat1 deletion were found to be circadian or non-circadian, targets or non-targets of paraspeckles, and to be associated with many key biological processes showing that Neat1, in interaction with the circadian system or independently, could play crucial roles in key physiological functions through diverse mechanisms.

Direct RNA-RNA interaction between Neat1 and RNA targets, as a mechanism for RNAs paraspeckle retention.

Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3'UTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA-RNA interactions. Using an RNA pull-down procedure adapted to select only RNAs engaged in direct RNA-RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5' end of Neat1 demonstrating that they are located in the shell region of paraspeckles.