Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Takeshi Yokoyama 0 Tsutomu Suzuki 0 0 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain ("7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections. - Ribosomes translate genetic information encoded in mRNAs into a corresponding sequence of amino acids to form a protein. Ribosomes consist of large and small subunits, each of which is a ribonucleoprotein complex formed by rRNAs and ribosomal proteins. The rRNAs form the basic structure of the ribosome, and play central roles in the fundamental processes of protein biosynthesis. Recent structural studies of each subunit and of the 70S ribosome revealed that the functional cores subserving mRNA decoding and peptide-bond formation consist entirely of rRNAs, thus implying that the ribosome is an RNA-based machine (17). Although the functional regions of rRNAs are highly conserved, the architecture of rRNAs diversifies amongst organisms and organelles. In mammalian mitochondria, the lengths of rRNAs are shortened to approximately half that of prokaryotic rRNAs. Many helices in rRNAs are shortened or missing, whereas all functional domains are conserved. Large regions of missing RNA segments are replaced by enlarged ribosomal proteins and other, mitochondriaspecific proteins (813). In contrast, eukaryotic ribosomes contain elongated rRNAs and an increased number of ribosomal proteins (1417). It is thought that the architecture of rRNAs might have coevolved with ribosomal proteins so as to preserve the fundamental structure and function of ribosomes in all domains of life. Variations in the RNA-to-protein ratio found in ribosomes from various organisms indicate that some degree of architectural flexibility of is permissible in the evolutionary refinement of ribosomal structure. Compared to Escherichia coli 23S rRNA, with 2904 nt, yeast (Saccharomyces cerevisiae) 26S rRNA consists of 3392 nt, while human 28S rRNA consists of 5025 nt (15). The additional residues in eukaryotic rRNAs are inserted at several specific sites in the secondary structures of prokaryotic rRNAs as expansion segments (ESs) (15). ESs vary in their size and sequence from species to species. ESs are categorized as 12 distinct segments (designated es1 to es12) in the small subunit rRNAs, and 41 distinct segments (designated ES1 to ES41) in the large subunit rRNAs (Figure 5A and B). As ESs can be found in nonconserved regions of rRNAs, it is thought that ES insertion does not disturb the fundamental function of rRNAs (15). ESs are known to contact with other ESs to form a large structural element of eukaryotic ribosomes (1821). The structural diversity conferred upon eukaryotic ribosomes by ESs affects the complex regulatory mechanism of eukaryotic translation (14,22,23). Although the exact functions of ESs in rRNAs remain elusive, cryo-EM studies of eukaryotic ribosomes are providing clues revealing some of the functional aspects of ESs. ESs provide sites for eukaryote-specific intersubunit bridges, as well as scaffolds allowing additional proteins to bind to ribosomes (14). It has been revealed that ES24 near Helix 59 in the large subunit of the yeast 80S ribosome interacts directly with the Sec61 complex. This interaction suggests that ES24 plays an important role in the process of cotranslational protein translocation, by serving as an attachment site for the protein-conducting channel in endoplasmic reticulum (22). Upon binding of Sec61, ES27, an essential rod-like component, drastically changes its conformation, moving from the peptide exit site to a site close to L1 stalk. It has been proposed that movement of ES27 coordinates access of non-ribosomal protein factors to the peptide exit channel (22). In the protozoan Trypanosoma cruzi 80S ribosomes, es6 and es7 in the small subunit form a large domain which might assist in escorting mRNAs to the ribosome (19). The availability of comparative and phylogenetic analyses of rRNA sequences with secondary structures provides us with many insights into the functional and structural evolution of rRNAs, whereas a purely experimental approach to investigating rRNA evolution is limited. Genetic insertion of short RNA segments into rRNAs is possible however, and allows us to probe ribosome architecture and function. Earlier experiments employing genetic insertion of RNA segments into rRNAs used cryo-electron microscopy to identify the placement of each rRNA helix within the ribosome structure (24,25). A 17-nt segment or a tRNA-like element was introduced at several positions of 23S rRNA by a conventional mutagenesis approach, and extra electron densities corresponding to the insertions were then observed. To examine the architectural evolution of rRNAs in an empirical and unbiased manner, it is necessary to design and adhere to a specific method for distinguishing functional insertions in rRNAs from a large number of random genetic insertions. Here, we describe a systematic genetic approach for selecting functional rRNA variants bearing short, inserted RNA segments. We previously developed a comprehensive genetic selection method which we named systematic selection of functional sequences by enforced replacement (SSER) (26). This method allowed us to rapidly identify residues and sequences essential for ribosome function in E. coli cells, from randomized rRNA libraries. We employed this approach to analyze the peptidyltransferase center (26), the conserved loop sequence of H69 (27) and the internal bulge sequence of H66 for the L2 binding site (28). For the current analysis, we constructed an rRNA library by randomly inserting a short RNA segment using a Tn5 transposon, and then subjected the library to SSER to isolate rRNA variants with functional insertions. To be identified as functional, the activity of the inse (...truncated)