High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast

Abstract

While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein–rRNA interactions, providing intriguing insights into the temporal order of protein–rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.

Pol5 is an essential ribosome biogenesis factor required for 60S ribosomal subunit maturation in Saccharomyces cerevisiae

“…Recent cryo-EM analyses of early pre-60S r-particles indicate that by the time most B-factors appear to be stably bound, the six 25S/5.8S rRNAs domains have been folded but not fully compacted (Kater et al 2017;Sanghai et al 2018;Bassler and Hurt 2019;Klinge and Woolford 2019). These results are consistent with those obtained by studying the composition of purified pre-60S r-particles containing progressively 3'-elongated fragments of 27S pre-rRNA (Chen et al 2017;Chaker-Margot and Klinge 2019) and those of high-throughput RNA structure probing analyses on purified nucleolar pre-60S r-particles (Burlacu et al 2017). Regarding r-proteins, several 60S r-subunit proteins, including uL6, uL22, eL19, uL14, uL23, uL24, eL27, eL34, uL29, and eL37 (formerly L9, L17, L19, L23, L25, L26, L27, L34, L35, and L37, respectively), have been reported to be more or less important for efficient conversion of 27SB into 7S and 25.5S pre-rRNAs (see de la Cruz et al 2015 and references therein).…”

The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor

“…Npa1p was also found cross-linked to two sites within ITS2 ( Fig 9A ). In the proposed “ring-pin” model of ITS2 secondary structure [ 7 ], Npa1p is positioned at the base and in the central region of the hairpin structure ( Fig 9A ), where no AF binding had so far been reported.…”

“…Within the nascent pre-60S particle, methylation and pseudouridylation of specific residues of 25S rRNA take place, carried out by snoRNPs. In addition, the 25S and 5.8S rRNAs and flanking sequences start folding [ 7 ]. In the mature 60S subunit, the 25S rRNA is folded in 6 structural domains (I to VI), each beginning with a root helix.…”

dStruct: identifying differentially reactive regions from RNA structurome profiling data

“…In fact, recent developments have led to a diversity of structure probing or structure profiling (SP) technologies [4,5]. These technologies have made it possible to perform comparative analysis of structures of select RNAs or whole RNA structuromes simultaneously [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

“…Besides this, PARCEL does not account for coverage variations within a transcript. Second, in many studies, candidate regions, which might be DRRs, are not known a priori [8,11,18]. Hence, they need to be constructed de novo.…”