rRNA expansion segment 7 in eukaryotes: from Signature Fold to tentacles

Abstract

The ribosomal core is universally conserved across the tree of life. However, eukaryotic ribosomes contain diverse rRNA expansion segments (ESs) on their surfaces. Sites of ES insertions are predicted from sites of insertion of micro-ESs in archaea. Expansion segment 7 (ES7) is one of the most diverse regions of the ribosome, emanating from a short stem loop and ranging to over 750 nucleotides in mammals. We present secondary and full-atom 3D structures of ES7 from species spanning eukaryotic diversity. Our results are based on experimental 3D structures, the accretion model of ribosomal evolution, phylogenetic relationships, multiple sequence alignments, RNA folding algorithms and 3D modeling by RNAComposer. ES7 contains a distinct motif, the 'ES7 Signature Fold', which is generally invariant in 2D topology and 3D structure in all eukaryotic ribosomes. We establish a model in which ES7 developed over evolution through a series of elementary and recursive growth events. The data are sufficient to support an atomic-level accretion path for rRNA growth. The non-monophyletic distribution of some ES7 features across the phylogeny suggests acquisition via convergent processes. And finally, illustrating the power of our approach, we constructed the 2D and 3D structure of the entire LSU rRNA of Mus musculus.

Figure 1.

Secondary structures of two eukaryotic LSU rRNAs. (A) S. cerevisiae, and (B) H. sapiens. rRNA of ribosomal common core as defined in (2) is black. ESs are highlighted in teal and labeled according to Gerbi (8).

Figure 2.

Secondary structures of ES7 mapped onto the canonical eukaryotic tree of life. Basal Helix 25 (boxed in the center) is part of the universal core; it is shown in blue in secondary structures as well as in the inset of H. sapiens ES7. Green indicates the ES7 Signature Fold, which is universal to rRNA of all eukaryotes. Yellow is universal to metazoans. Red is metazoan specific rRNA extensions, some of which reach extreme lengths in birds and mammals (tentacles). Colors of branches and internal nodes represent allocation of species (leaves) into Groups 0–3. The GC content (shown as percentage) for each ES7 is displayed next to each leaf of the tree. The asterisks indicate related species (D. melanogaster and A. albopictus) with a highly divergent GC content. Inset: the structure of ES7 of H. sapiens with the Signature Fold in bold green and blue. Helices labeled according to H. sapiens schema (4). All secondary structures in this document were visualized with the web portal RiboVision (34). The 3D structure of a prokaryotic ribosome with H25 highlighted in the blue box. In this image, colored by distance from the functional centers (peptidyl transferase center in the LSU and decoding center in the SSU) the ribosomal proteins are in gray.

Figure 3.

A schematic of the work-flow used to model 2D and 3D structures of eukaryotic rRNAs. RC indicates automated steps performed by RNAComposer.

Figure 4.

The ES7 Signature Fold. (A) Secondary structure. (B) Three-dimensional structure. The coloring scheme is the same as in Figure 2. In panel (B), all experimental and modeled structures from Table 1 are superimposed.

Figure 5.

Conservation (GASE) and co-variation (PASE) scores mapped onto the secondary structures of ES7 from Groups 1–3. (A) GASE and (B) PASE computed from ES7 MSA of Group 1 and mapped onto ES7 of S. cerevisiae; (C) GASE and (D) PASE computed from ES7 MSA of Group 2 and mapped onto ES7 of D. melanogaster; (E) GASE and (F) PASE computed from ES7 MSA of Group 3 and mapped onto ES7 of H. sapiens. Both scores range from 0 (dark blue, absolute conservation or co-variation) to 2 (yellow, random signal, no conservation or covariation). Intermediate values are indicated by the color bar. The ES7 MSAs of Groups 1–3 are given in Supplementary Data Set 4.

Figure 6.

RNA accretion in ES7, based on comparison of experimental 3D structures and their evolutionary relationships. The lineage leading to H. sapiens (Metazoan) is highlighted. The rRNA at each ancestral node, highlighted by colored circles, is approximated by rRNA that is common to daughters (2). rRNA is depicted schematically, in secondary structures, and in three dimensional structures. A stem loop of H25 (E. coli) establishes the base of ES7. An expansion of the apical loop extends H25 in H. marismortui; a bulge expands into an internal loop in P. furiosus, which extends into a new expansion ES7a in T. thermophilia; this element undergoes further adjustments in H. sapiens. PDB IDs of the source structures are indicated below the 3D structures. Chain IDs and nucleotide numbers are given in Supplementary Table S3. The 3D structures are compiled in Movie 2.

Figure 7.

A three-way junction emerges from a helix through a series of elementary steps of rRNA accretion. Existing and emerging elements are depicted by their secondary and 3D structures and by schematic representations. (A) Double helical region; (B) 1 nt bulge; (C) 6 nt bulge; (D) 1 and 6 nt loop; (E) 3 and 6 nt loop; (F) three-way junction. This trajectory is based on experimental 3D structures that are ordered by size. The evolutionary relationship is inferred. PDB IDs of the source structures are indicated below the structures. Chain IDs and nucleotide numbers are given in Supplementary Table S4.

Figure 8.

Secondary structure of the LSU rRNA of M. musculus. Expansion segments are outlined.