Assembly and early maturation of large subunit precursors

Abstract

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As preribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification, and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors, but how these particles emerge is still unknown. Here, we use the expression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II, and VI of the assembling 25S rRNA.

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).…”

Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae

“…Ribosome assembly begins in the nucleolus with the synthesis of the primary 35S rRNA transcript and the 5S rRNA. The primary transcript contains the 18S, 5.8S, and 25S rRNAs and four spacer regions that are removed during ribosome assembly: two external transcribed spacers (ETS) and two internal transcribed spacers (ITS) (S1 Fig) . The r-proteins and the biogenesis factors assemble on the rRNA in a hierarchical order [6][7][8][9][10]. While most of the biogenesis factors promote the correct architecture of the subunits by chaperoning and modifying the rRNA, others drive structural rearrangements and removal of the spacer regions.…”

Autoregulation of yeast ribosomal proteins discovered by efficient search for feedback regulation

“…We are particularly interested in finding new examples of post-transcriptional autoregulation of protein expression so our initial focus is on ribosomal proteins, which are commonly translationally autoregulated in bacteria but 27 , 28 , 49 , 50 for which many fewer examples are known in yeast. Ribosome synthesis in yeast is subject to feedback regulation in part by alternative functions of ribosomal proteins 51 – 55 . There are several examples of post-transcriptional autoregulation by yeast ribosomal proteins, most often through inhibition of splicing necessary for protein expression.…”