Abstract
SUMMARYRoles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units positioned across multiple loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. It remains elusive whether expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may have functional roles in translation control. Here, we develop variable expansion segment-ligand chimeric ribosome immunoprecipitation RNA sequencing (VELCRO-IP RNA-seq), a versatile methodology to generate species-adapted ESs and to map specific mRNA regions across the transcriptome that preferentially associate with ESs. Application of VELCRO-IP RNA-seq to a mammalian ES, ES9S, identified a large array of transcripts that are selectively recruited to ribosomes via an ES. We further characterize a set of 5′ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome binding. Thus, we present a technology for studying the enigmatic ESs of the ribosome, revealing their function in gene-specific translation.
Highlights
The ribosome is life’s most ancient molecular machine, with an RNA structural core that is universally shared across all species
We have previously discovered that the mammalian ES9S in 18S ribosomal RNA (rRNA) directly interacts with a 50 untranslated region (UTR) RNA element in a Homeobox (Hox) transcript to promote translation initiation of the Hox mRNA in a transcript-specific manner
Engineering of yeast ribosomes with customized rRNA expansion segments (ESs) for VELCRO-IP When the secondary structures of 18S rRNAs for evolutionarily distant baker’s yeast (S. cerevisiae) (Armache et al, 2010) and human (H. sapiens) (Natchiar et al, 2017) are compared (Figures 1A and S1A–S1C), the basal stem region of helix h39 adjacent to ES9S is highly conserved, whereas the distal portion of ES9S is highly variable in length, structure, and sequence (Figures 1A– 1C, boxed region)
Summary
The ribosome is life’s most ancient molecular machine, with an RNA structural core that is universally shared across all species. The human ribosome is 1 MDa larger than the yeast ribosome, which in turn is another 1 MDa larger than the bacterial ribosome. This is largely because of the insertions of blocks of sequences called expansion segments (ESs) as they expand the eukaryotic ribosomal RNA (rRNA): the longest ESs are more than 700 nt in Homo sapiens (H. sapiens) and resemble flexible tentacles that extend from the ribosomal surface (Anger et al, 2013; Armache et al, 2010; Gerbi, 1996). ESs are located in rRNA regions of lower primary sequence conservation, which initially suggested that they are neutral mutations that do not interfere with essential rRNA functions in protein synthesis across all kingdoms (Gerbi, 1986)
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