Profusion of G-quadruplexes on both subunits of metazoan ribosomes

Abstract

Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadruplexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.

Human Ribosomal G-Quadruplexes Regulate Heme Bioavailability

“…Immunofluorescence experiments with BG4 and L19 antibodies suggest a specialized fraction of cytosolic ribosomes (~5%) form G4s and that most extra-nuclear G4s (~83%) are ribosomal. The small fraction of ribosomes observed to form G4s in vivo contrasts with the high stability of ribosomal G4s in vitro (7,8). This difference is consistent with Guo and Bartel, who propose that eukaryotic cells have a robust machinery that unfolds G4s (16).…”

“…We previously reported that surfaces of both the SSU and the LSU contain G4 sequences (7,8). A broad variety of data are consistent with more extensive formation of G4s on the LSU than on the SSU.…”

“…BioTASQ captures G4s. We previously used BioTASQ to demonstrate that human rRNA forms G4s in vitro (Figure 2E) (8). Here, we captured rRNA G4s from crosslinked HEK293 cells by methods summarized in Figure 2F.…”

“…rRNA oligomers GQES7-a ( Figure 3A (7). GQes3 is a fragment of expansion segment 3 of human SSU rRNA (8). Each of these oligonucleotides is known to form G4s and each caused a pronounced increase in the Soret band of hemin at 400 nm.…”

“…G4s are nucleic acid secondary structures that are composed of four guanine columns surrounding a central cavity that sequesters monovalent cations. Our hypothesis is based on the high affinity of heme for G4s (KD ~10 nM) (4)(5)(6), our work demonstrating that rRNA forms extensive G-tracts in vitro (7,8), the extreme stabilities of rRNA G4s in vitro (7,8) and the extraordinary abundance of rRNA in vivo (9). DNA G4s are proposed to help regulate replication (10), transcription (11), and genomic stability (12).…”