rG4detector, a novel RNA G-quadruplex predictor, uncovers their impact on stress granule formation

Abstract

RNA G-quadruplexes (rG4s) are RNA secondary structures, which are formed by guanine-rich sequences and have important cellular functions. Existing computational tools for rG4 prediction rely on specific sequence features and/or were trained on small datasets, without considering rG4 stability information, and are therefore sub-optimal. Here, we developed rG4detector, a convolutional neural network to identify potential rG4s in transcriptomics data. rG4detector outperforms existing methods in both predicting rG4 stability and in detecting rG4-forming sequences. To demonstrate the biological-relevance of rG4detector, we employed it to study RNAs that are bound by the RNA-binding protein G3BP1. G3BP1 is central to the induction of stress granules (SGs), which are cytoplasmic biomolecular condensates that form in response to a variety of cellular stresses. Unexpectedly, rG4detector revealed a dynamic enrichment of rG4s bound by G3BP1 in response to cellular stress. In addition, we experimentally characterized G3BP1 cross-talk with rG4s, demonstrating that G3BP1 is a bona fide rG4-binding protein and that endogenous rG4s are enriched within SGs. Furthermore, we found that reduced rG4 availability impairs SG formation. Hence, we conclude that rG4s play a direct role in SG biology via their interactions with RNA-binding proteins and that rG4detector is a novel useful tool for rG4 transcriptomics data analyses.

Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

“…1 This means that any rG4 probes must exhibit exceptionally high binding selectivity for G4s. Additionally, as rG4s are often found in phase-separated regions of the cell (and may even drive phase separation events), [60][61][62] probe uptake must be considered when interpreting fluorescence images. For example, differences of probe uptake into phase-separated organelles confound measurements of fluorescence intensity, where increased fluorescent signal may be due to local probe concentration rather than G4 abundance.…”

Small-molecule-based regulation of gene expression in human astrocytes upon both positive and negative actuations of G-quadruplex control systems

“…Interestingly, the molecular components that are most affected by both PDS and PhpC are associated to stress granule (7/84, 8%, s = 1.25) and polysome formation (5/65, 8%, s = 1.22), fully in line with the pivotal role play by G4s in response to cellular stress and in translation. [65][66][67] It is thus worth noting that both PDS and PhpC strongly impact RNA metabolism, although through different angles. PDS triggers a scatter-gun type of damage, affecting most if not all cellular circuities, which is in line with the RNA-seq analysis: indeed, the 354 PDStranscripts were found to belong to dozens of different cellular pathways (including DNA repair (62/497, 13%), replication (51/203, 25%) and unwinding (25/89, 28%), etc.)…”

Visualizing liquid-liquid phase transitions

“…These repeat sequences are known to form RNA G-quadruplex structures. 39,40 To get a preliminary impression if SERF2 has some specificity in binding these non-canonical structures, we tested its interaction with two RNAs with similar sequences: (AGG)5, which folds into a rG4 quadruplex, and (ACG)5, that does not (Figure S3C). 40 SERF2 binds to (AGG)5 with a binding affinity (K D ) of 1.8 ± 0.5 µM, but shows no measurable binding to (ACG)5 (Figure S3C).…”