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\DOI{10.5802/crbiol.181}
\datereceived{2025-05-06}
\daterevised{2025-06-23}
\dateaccepted{2025-07-01}
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\dateposted{2025-09-16}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Structure of the ribosome from highly developed organisms. Mini
review}

\alttitle{Structure du ribosome d'organismes hautement
d\'{e}velopp\'{e}s. Mini revue}

\author{\firstname{Liliia} \lastname{Nurulina}}
\address{Institute of Fundamental Medicine and Biology, Kazan Federal
University, Kazan, Russia}

\author{\firstname{Salvatore} \lastname{Terossu}}
\address{Integrated Structural Biology, IGBMC -- IGBMC -- CNRS UMR 7104
-- Inserm U 1258, France}

\author{\firstname{Marat} \lastname{Yusupov}\CDRorcid{0000-0001-5544-0597}\IsCorresp}
\addressSameAs{2}{Integrated Structural Biology, IGBMC -- IGBMC -- CNRS UMR 7104
-- Inserm U 1258, France}
\email[M. Yusupov]{marat@igbmc.fr}

\shortrunauthors

\keywords{\kwd{Cryo-EM}\kwd{RNA extension
segments}\kwd{Structure}\kwd{Chick embryo}\kwd{Ribosome}}

\altkeywords{\kwd{Cryo-EM}\kwd{Segments d'expansion de
l'ARN}\kwd{Structure}\kwd{Embryon de poussin}\kwd{Ribosome}}

\begin{abstract}
Ribosomes of highly evolved organisms are larger than their bacterial
counterparts not only in terms of the number of proteins but also in
terms of RNA length extensions. Some extensions do not fold into the
three-dimensional structure of the ribosome in cryo-EM analysis and
cannot be crystallized for X-ray examination. These expansion segments
are not visible due to flexibility. In this mini-review, we propose an
approach to study the chick embryo ribosome as an example of a highly
evolved organism to visualize the expansion segments and study the
mechanism of in situ tetramerization and crystallization of ribosomes.
\end{abstract}

\begin{altabstract}
Les ribosomes des organismes hautement \'{e}volu\'{e}s sont plus gros
que leurs homologues bact\'{e}riens non seulement en termes de nombre
de prot\'{e}ines, mais \'{e}galement en termes d'extensions de longueur
d'ARN. Certaines extensions ne se replient pas dans la structure
tridimensionnelle du ribosome dans l'analyse cryo-EM et ne peuvent pas
\^{e}tre cristallis\'{e}es pour l'examen aux rayons X. Ces segments
d'expansion ne sont pas visibles en raison de la flexibilit\'{e}. Dans
cette mini-revue, nous proposons une approche pour \'{e}tudier le
ribosome de l'embryon de poussin comme exemple d'organisme hautement
\'{e}volu\'{e} afin de visualiser les segments d'expansion et
d'\'{e}tudier le m\'{e}canisme de t\'{e}tram\'{e}risation et de
cristallisation in situ des ribosomes.
\end{altabstract}

\maketitle

{\vspace*{2pt}}

\twocolumngrid

\end{noXML}

\section{Introduction}

Ribosomes are multi-megadalton cellular machineries constituted of
proteins and rRNA in charge of protein biosynthesis. Throughout
evolution, ribosomes have increased in size. Expansions occur both at
the protein level, with the enlargement of all large ribosomal
proteins, and at the RNA level---with the increase in
length of ribosomal RNA. The expansions within ribosomal RNA clusters
in conserved regions facing the periphery of the ribosome. Recent
biochemical and structural studies have shed light on how specific rRNA
extension segments (ESs) interact with protein factors during
translation. High-resolution crystal structures of bacterial and yeast
ribosomes (Figure~\ref{fig1}), in combination with more recent cryo-EM analyses,
have been crucial in elucidating the fundamental mechanisms of protein
synthesis in prokaryotic and eukaryotic systems \citep{1,2,3}.
However, after 35 years of ribosome structural studies, there is little
structural information about some ribosomal elements such as the
L7/L12-stalk (P-stalk for eukaryote), the protein pentameric complex
and several rRNA expansion segments (ES) of highly developed organisms
\citep{1,4,5}. 

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Crystal structures of bacteria~70S and yeast~80S
ribosomes with molecular weight according to 2.3 and 3.3~MDa.
Cryo-EM structure of the human~80S ribosome, as an example of the
ribosome from highly developed organisms with a molecular weight of
4.3~MDa.}
\end{figure*}

A genome sequence comparison of ribosomal RNA from different kingdoms
indicates an extension of the rRNA as the complexity of the organisms
increases \citep{6,7}. Among the eukaryotes, the mass of the
ribosome varies from 3.3~MDa in unicellular eukaryotes to 4.3~MDa in
humans (Figure~\ref{fig1}). This difference is mainly due to the
enlargement of four specific expansion segments distributed across the
rRNA \citep{8}. The expansion segments are generally located at the
solvent surface and do not directly interact with ribosome functional
sites \citep{6,9,10}. Even though the locations of the insertion
sites of the expansion segments coincide, their length and nucleotide
composition differ among eukaryotes: ESs are generally more GC-rich in
vertebrates than in invertebrates and unicellular eukaryotes
\citep{6,7}.

Structurally, rRNA expansion segments can be divided into two clusters:
the first type is known to interact with ribosomal proteins or rRNA,
such as ES31L and ES39L (Figure~\ref{fig4}); the second type of rRNA ES
includes long rRNA helices anchored to the ribosome via their bases.
ESs belonging to this group can adopt different conformations, as
observed for the flexible ES6S and ES27L, although their functional
role is not yet clear \citep{1}. Studies have been carried out to
investigate the involvement of ESs in translational fidelity, ribosome
biogenesis, and protein folding \citep{8}. ES6S, \mbox{located} in the
vicinity of the mRNA entry and exit sites, and ES27L, localized close
to the polypeptide exit tunnel, were shown to help translation factors
access the ribosome \citep{11,12}. Using systematic deletion
experiments of specific ESs, it was shown that almost all ESs in
\textit{Saccharomyces cerevisiae} are required and vital for ribosome
biogenesis \citep{13}. Also, it was demonstrated that ES27L and ES7L
orchestrate the binding and anchoring of the acetyltransferase NatA
complex, which is required for acetylation of the nascent peptide chain
and facilitates binding of the conserved enzyme methionine
aminopeptidase (MetAP) responsible for cleaving off the N-terminal
methionine residue \citep{14,15}. Generally, ESs are believed to be
involved in many different processes that maintain ribosome efficiency
\citep{8}.

An interesting observation was made while comparing the sedimentation
coefficients of bacterial, yeast and human ribosomes. Comparison of the
sedimentation coefficients of the bacterial ribosomal 50S and 30S
subunits, the bacterial 70S ribosome, the yeast 80S ribosome and the
human ribosome showed a partially unfolded structure of the human
ribosome. The experimental sedimentation coefficients of the human
ribosome and the yeast ribosome were 78S and 80S, respectively
(Figure~\ref{fig2}). However, the molecular weight of the human
ribosome is approximately 1,000,000~Da more than the yeast ribosome
owing to the longer expansion segments of the 28S RNA. A partially
unfolded structure of the\break human \mbox{ribosome} expansion segments made the
crystallization of this ribosome difficult and made other structure
functional studies challenging to accomplish.

\begin{figure}
\includegraphics{fig02}
\caption{\label{fig2}Dependence of the sedimentation coefficient of the
ribosome particle on molecular weight. Graphic kindly provided by S.
Agalarov.}
{\vspace*{-3pt}}
\end{figure}

Most studies regarding the function of expansion segments have been
carried out in yeast, whose expansion segments are shorter than in
mammals \citep{11}. The main challenges in answering these enduring
questions lie in the different intrinsic limitations of structural
techniques, such as the need for crystal growth in crystallography and
the limited resolution caused by flexibility and averaging in cryo-EM
and cryo-ET. The rRNA expansion segments and protein elements mentioned
above are highly flexible and often unstructured, which \mbox{hinders} both
proper crystallization---crucial for achieving
high-resolution data---and accurate image alignment in
cryo-EM and cryo-ET, essential for resolving fine structural details
\citep{6,7,16}. A successful approach to visualize expansion
segments is cryo-ET in situ, which has been used efficiently to resolve
ES of \textit{Dictyostelium discoideum} ribosomes. Despite the low
resolution achieved, Hoffmann and collaborators depicted the
three-dimensional features of rRNA ES in a near-physiological
environment, capturing them in complex with several translation
factors. Their structural work supported the long-standing hypothesis
behind the role of ES in translation fidelity and its fine-tuning, thus
suggesting their transversal role in the evolution of higher eukaryotes
\citep{17}. An alternative way to investigate the structure of
these flexible regions involves a more biological rather than technical
approach. Starting from functional data, researchers made the
hypothesis that some protein factors could lock such regions in a
certain conformation, allowing their visualization. It is the case of
the maturation protein Arx1, which interacts with ES27L in the pre-60S
particles of \textit{S.~cerevisiae} ribosomes. The interaction with
Arx1 stabilizes the region, making the ES27L flexible helix a more
stable hub for further protein interactions that would lead to the
export of the pre-60S ribosomes to the cytosol \citep{18}.

\section{Natural crystals of ribosomes of highly\unskip\break developed
organisms}

An important observation, made several decades ago, was the
identification of ribosome hibernation in \textit{Gallus gallus}.
During study of the hypothermic effects on chicken microtubules, highly
ordered bidimensional layers of ribosomes were discovered
\citep{21,19,20}. Bidimensional layers were studied using electron
diffraction techniques, thus demonstrating their crystalline nature,
with each unit cell containing four ribosomes---tetramers
\citep{22}. Tetramers were found and isolated from all tissues of
the embryo, demonstrating that under cold exposure chicken ribosomes
can exist in various forms: monomeric, tetrameric, and in crystalline
layers \citep{19,23}. Several questions regarding the formation,
the structure and the role of these tetramers and larger aggregates
remain unanswered. Due to the compact form of both layers and
tetramers, it is easy to think that expansion segments and protein
factors might play a role in the formation of ribosomal sheets, by
stabilizing ribosomes via rRNA--rRNA and/or rRNA--protein interactions.
The study of this phenomenon began in the early 70s, but due to a lack
of technical capabilities, no certain results have been obtained.

Ribosome crystalline layers have been found in sand lizard
(\textit{Lacerta agilis}) and common chicken eggs (\textit{Gallus
gallus}) exposed to cold stress \citep{24,25}. We can conclude that
this is a physiological response occurring under conditions of
stress-induced hypothermia. The ability of the ribosomes from chick
embryos to form tetramers and 2-D crystals {in situ} under
stress conditions allows its purification and recrystallization
\citep{23}. Isolated \textit{G.~gallus} ribosomes could be used for
the stabilization and structural study of expansion segments.

\section{\textit{Gallus gallus} ribosomes for structural study.
Possible stabilization of expansion segments}

As a starting point of our research, the recently structure of isolated
\textit{G.~gallus} 80S ribosomes (Figures~\ref{fig3} and~\ref{fig4}) has
been determined by a cryo-EM study \citep{5}. 

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Structure of \textit{G.~gallus} ribosomal 60S
subunit. Ribosomal RNA is shown in grey and ribosomal proteins in
color. The left side represents the ribosomal interface, while the
right side represents the ribosomal solvent side.}
\end{figure*}


\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Structure of the \textit{G.~gallus} ribosomal 40S
subunit. Ribosomal RNA is shown in grey and ribosomal proteins in
color. The left side represents the ribosomal interface, while the
right side represents the ribosomal solvent side.}
{\vspace*{-1pt}}
\end{figure*}

As results all ribosomal proteins have been modeled in the structure
with the only exception of P1 and P2 proteins of the P-stalk of the
large ribosomal subunit (Figure~\ref{fig3}). This protein complex is
flexible and not visible in cryo-EM structures and crystal structures
of \textit{S.~cerevisiae} ribosomes as well \citep{10,26}. 

\begin{figure*}
{\vspace*{-3pt}}
\includegraphics{fig05}
{\vspace*{-3pt}}
\caption{\label{fig5}Secondary structure of 28S rRNA: on the left, the
secondary structure from \textit{Gallus gallus}; and on the right, the
one from \textit{Homo sapiens}. In black are the rRNA conserved between
the two species. Expansion segments are represented in yellow/red
color. Rred indicate the parts of expansion segments which are flexible
and not visible in cryo-EM three-dimensional structure.}
{\vspace*{-3pt}}
\end{figure*}
 
The interface between ribosomal subunits, as well as the area
surrounding the mRNA entrance and the polypeptide exit tunnel, is
highly conserved and contains very few expansion segments and
eukaryote-specific proteins. The ribosomal RNA expansion elements are
located predominantly on the periphery of the solvent-exposed sides of
both subunits of the eukaryotic ribosome.

The insertions of expansion segments in the sequence of the rRNA of
\textit{G.~gallus} is conserved among other eukaryotic organisms.
However, the length of these inserted ES differs between species, and
some features of the expansion segments are specific to avians
(Figure~\ref{fig5}). Our map revealed differences in the structural
arrangement of expansion segments in the 28S rRNA of \textit{Gallus
gallus}. The main differences, when compared to its human counterpart,
were observed in the highly variable expansion segment ES7L and between
the interaction patterns of the expansion segments ES15L, ES10L, ES9L
and ES7L. These differences suggest that the packaging of expansion
segments may vary from species to species. The ES7L tentacle was found
to be longer than in other species, and one of our assumptions was that
this tentacle could participate in the formation of tetramers and
aggregates (Figure~\ref{fig5}). The solved structure of the 80S
\textit{G.~gallus} ribosome can be further utilized while working on
the structure of the ribosomal tetramers and aggregates.

\section{Conclusion}

We suggest that the stabilization of chick embryo ribosomes is possible
in an environment similar to stress cold conditions {in vivo}.
It is impossible to imagine the crystallization of ribosomes containing
unfolded expansion segments into 3-dimensional crystals, as was shown
earlier \citep{23}. We conclude that the behavior of human ribosome
during sedimentation experiments is the result of partial unfolding of
ribosomal RNA during purification and sample preparation. Similar
results of unfolded expansion segments were obtained in all cryo-EM
studies of ribosomes from highly developed organisms including human
and chick embryos \citep{5,6,7}. Therefore, it is becoming clear that
some sort of stabilization is needed to decipher the ES spatial
disposition. Finding the right model to address these structural
questions about the role of ES is of paramount importance to dive
deeper in the \mbox{understanding} of translation regulation at the molecular
level. For this reason, we suggest using {in situ} cryo-ET to
study ribosomal tetramers and aggregates that are stabilized under cold
stress conditions. This could help resolve their three-dimensional
structure, elucidate the mechanism of aggregate formation, and
visualize expansion segments during ribosomal function or
intermolecular contact formation.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that  could benefit from this article, and have
declared no affiliations other than their research organizations.

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