G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA

Abstract

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

nearBynding: A flexible pipeline characterizing protein binding to local RNA structure

“…Indeed, we observed strong RBP binding at G4s for multiple published G4-binding proteins such as NONO, FUS, GRSF1, DROSHA, and DDX42 ( Fig. 5A and S6A) [40][41][42][43][44]. Additionally, many of the RBPs that exhibited the strongest G4-binding signal-PRPF4, GTF2F1, FAM120A, CSTF2T, and DDX6-have recently been shown to bind at putative G4 sites in mRNA UTRs [45].…”

G-Quadruplexes in RNA Biology: Recent Advances and Future Directions

“…In particular, we discuss the transient nature of RG4s and outline the mechanistic link between RG4s and RNA modifications, mitochondria, phase transitions, and immune evasion. This will be implemented through an analysis that intersects proteomics data identifying RBPs [13][14][15][16][17] with the most recent data sets on RG4-binding factors [18][19][20][21][22][23][24][25].…”

“…However, in vivo, since mRNAs must be unfolded to fulfill their messenger function, they are challenged by cellular components that convert them to either a linear form or an alternative Watson-Crick base pair structure. In view of their abundance and their ability to regulate all post-transcriptional gene expression steps, RBPs emerged as leading candidates to regulate RG4 folding in cellulo, paving the way for several studies that characterized the RG4-binding protein machinery using unbiased RNA affinity proteomic-wide approaches (termed RNA purification coupled with mass spectrometry, RP-MS) [18][19][20][21][22][23][24][25] and/or in silico analysis of the association between RG4-forming sequences and RBP-binding sites [5,33]. These studies, together with recent in cellulo RG4-capturing approaches [34] and live-cell imaging of RG4 folding and unfolding [35], provided evidence of transient RG4 folding while reinforcing the view that some RBPs play a role in shifting RG4s toward an unfolded state.…”

“…Recent findings suggesting that almost all RG4s exist in unfolded conformations in cellulo [3] created a debate regarding their existence in living cells. However, their visualization using antibody-based [26] or small molecule-based [28,34,35] characterization dampened the skepticism and favored a model of a dynamic RG4 folding controlled by a protein machinery whose characterization constitutes an emerging challenge in the field [18][19][20][21][22][23][24][25]. The RG4 folding equilibrium is viewed as an on-off switch tuned toward a folded state by stabilizing cations, RG4-binding proteins, or RG4-stabilizing ligands (e.g., Phen-DC3, BRACO-19, or cPDS) [9].…”

A Sequence determinant in 3’UTR of mRNAs for Nuclear Retention by Paraspeckles

“…Thus, Neat1 can regulate expression of genes transcriptionally by sequestering proteins involved in the regulation of gene promoters, and co-transcriptionally by binding to mRNAs. While some studies have been undertaken to determine the sequence elements in Neat1 responsible for the recruitment of proteins(23), little is known about potential specific elements in paraspeckle mRNA targets except that several mRNA targets contain inverted repeats derived from SINE repeats in their 3'…”