The Expanding Riboverse: Comparison
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Mammalian ribosomes are 80S particles consisting of 80 ribosomal proteins (RPs) and 4 ribosomal RNA (rRNA) molecules in an approximately 1:1 mass ratio. 

  • ribosome
  • heterogeneity

1. Introduction

Subverting the conventional concept of “the” ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality than has traditionally been thought possible. A diverse array of researchers is collectively illuminating a universe of heterogeneous and adaptable ribosomes harboring differences in composition and regulatory capacity: These differences enable specialization. The expanding universe of ribosomes not only comprises an incredible richness in ribosomal specialization between species, but also within the same tissues and even cells.

2. Mitoribosomes and Chlororibosomes

The vast majority of the human proteome is synthesized by cytoplasmic or endoplasmic reticulum-associated ribosomes. However, 13 proteins of the mitochondrial oxidative phosphorylation machinery are translated exclusively by mitochondrial ribosomes (mitoribosomes), assembled from special mitochondrial RPs (encoded by nuclear DNA) and rRNAs encoded in the mitochondrial genome. Mitochondria are generally believed to be descendants of an ancient bacterium that entered into a symbiotic relationship with a primordial single-cell organism. As a result, the 55S mitochondrial ribosomes both structurally and dimensionally more closely resemble ribosomes found in modern day bacteria, rather than those in eukaryotic cells. Mitoribosomes, however, display a 2:1 protein-to-rRNA mass ratio, much higher, than the 1:2 ratio in bacterial ribosomes. Mitoribosomes also replaced large parts of their non-core rRNA components with proteins as part of a devolutionary process, similar to observations of other symbionts and parasites [1]. A close examination of the mitoribosome structure reveals that the new proteinaceous component replaced rRNA around its periphery, creating an outer “shell” around the conserved catalytic rRNA core [2]. This suggests that these ribosomes developed a kind of “armor” to protect their catalytic rRNA cores from attack by reactive oxygen species, which are produced in abundance by the process of oxidative phosphorylation in mitochondria. Thus, researchers suggest that mitoribosomes may have become “specialized” to function in this particularly harsh environment.
Chloroplasts are also endosymbionts with their own genomes (plastome) and bacteria-like 70S ribosomes. While the general protein-to-rRNA mass ratio of chlororibosomes does not differ much from those in bacteria, they do contain unique rRNA features, five chloroplast-specific ribosomal proteins, and unique protein extension elements. These enable the specialized function of chlororibosomes by promoting the association of translation factors involved in light- and temperature-dependent control of plant protein synthesis [3][4]. These too may be considered to have become “specialized” to optimize protein expression in the unique environment of the chloroplast.

3. Extreme Ribosomes

The first atomic resolution structures were generated using ribosomes that are specialized to function in extreme environments [5][6][7]. The organisms from which they were purified, Thermus thermophilis and Haloarculum marismortui, evolved to thrive in environments of high temperature and in high osmolarity, respectively. Biochemically, such conditions tend to destabilize non-covalent interactions, particularly hydrogen bonds and salt bridges. Accordingly, the biomolecules synthesized by extremophiles have evolved to maximize stability, traits which make them ideal for crystallization studies. Thus, one may consider these ribosomes optimized to ideally function in their respective extreme environments. Given the ancient origins of the ribosome and its central role in biology, the idea that ribosomes can become environmentally specialized has profound implications for the field of astrobiology.

4. Ribosomes of Parasites

Parasites tend to be minimalists because a) their host organisms are able to meet most of the metabolic needs, and b) their requirements for small genomes that can be rapidly replicated. Their ribosomes also tend to follow the trend towards minimalization. For example, microsporidia are eukaryotic parasites that have successfully adapted to parasitize almost all animals. Their genomes have condensed to be the smallest known in the Eukaryota, and their mitochondria are rudimentary. A recent cryo-EM analysis of Vairimorpha necatrix revealed the smallest known eukaryotic cytoplasmic ribosome to date [8]. The rRNA from this species has been reduced to a functionally conserved core due to the loss or severe compaction of all of the eukaryote-specific expansion segments, and it lacks two eukaryote-specific ribosomal proteins, eL38 and eL41. Furthermore, this species lacks the 5.8 rRNA, whose core sequences have been fused with the large subunit rRNA to create a unique 23S rRNA species. Interestingly, these ribosomes also associate with MDF1 and MDF2, distinct dormancy factors that may allow these organisms to save energy by storing inactive, “hibernating” ribosomes when they are not needed for active protein synthesis, e.g., during the spore stage.
Trypanosomes comprise a genus of parasitic flagellated protozoa in the class Kinetoplastea, best known for causing a variety of infectious diseases, including sleeping sickness, cutaneous leishmaniasis, and Chagas disease. Unlike the microsporidia, which have minimized rRNA content, trypanosomal rRNAs have become enlarged, containing unusually large expansion segments, a large rRNA domain that is not found in other eukaryotes, and additional rRNA insertions [9]. Additionally, some of the ribosomal proteins contain unique extensions, which enable the formation of four inter-subunit bridges that are not observed in other eukaryotic ribosomes. Curiously, although the genomic rDNA genes encode the four rRNA species, the large subunit rRNA is cleaved into six unique pieces. The functional aspects of these unique features are currently unknown.
The Apicomplexia include the genus Plasmodia, best known as the parasites responsible for malaria. Interestingly, Plasmodium species carry two cytoplasmic ribosome variants with different rRNA compositions. One of these is expressed in the mosquito vector, and the other is present in the mammalian host, although both can simultaneously occur for limited periods of time [10]. Presumably, these maximize the ability of the organism’s ribosomes to function in the very different environments of the insect vector and human host.

5. The Expanding Universe of Ribosome Diversity

The number of known exoplanets is now in the thousands. Taking into account the possible variations in parameters such as mass, orbit, composition, and distance to its star or stars, the number of conceivable unique planets approaches infinity. In parallel, the large number of known and possible ribosomes might be thought of as a constellation of ribosomes, which researchers call the “ribo-system” (Figure 1).
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Figure 1. The ribo-system. Various types of ribosomes populate the known riboverse. Each type of ribosome is specialized for particular environments, which are represented by orbits in the above image. Each orbit corresponds to each ribosomal species’ “habitable zone”.
The rate of expansion in ouresearchers'  knowledge of the degree of heterogeneity among ribosomes is similarly expanding, representing an exciting field of research. In addition to the different ribosomal protein paralogs discussed above, the functional importance of differences in their post-translational modification is beginning to emerge.
Recent advances, particularly in single-cell sequencing and quantitative mass spectroscopy, are helping to bring the visible riboverse into sharper focus. In mammals, the observation of tissue-specific patterning defects in mice lacking the RPL38 gene and its linkage to defects in translation of specific homebox (HOX) mRNAs represented a seminal breakthrough because of its implications for ribosome specificity in developmental biology [11]. The demonstration of the importance of this protein for translation of IRES-containing mRNAs created a new paradigm with regard to ribosome-mediated control of gene expression [12]. However, the idea of generating heterogeneity through subtraction [13] is less appealing than showing specificity through substitution of one ribosomal protein variant for another, e.g., the Rpl3l case described above. Later, sophisticated proteomics analyses revealed non-stoichiometric levels of ribosomal proteins, their association with various classes of other proteins, and association with different transcript sub-pools, painting a picture of ribosomes specialized by the ability of intrinsic protein content to recruit specific trans-acting factors [14][15]. Similarly, the evolution of rRNA expansion segments has been found to provide new platforms for binding trans-acting factors required for recruitment of specific mRNA classes [16].
Ufmylation is a metazoan-specific post-translational modification in which UFM1 proteins are conjugated to particular ribosomal proteins. The finding that differences in ufmylation confers differences in specificity for trans-acting proteins suggests another route for specialization via differences in post-translational modification of ribosomal proteins [14]. Since then, evidence for such has steadily accumulated [17][18][19][20][21][22][23][24][25][26][27]. Post-transcriptional modification of ribosomal rRNAs presents a similar path to ribosome specialization [28][29]. In particular, the recent findings of variably methylated rRNA bases [30] and of changes in rRNA modification levels in response to external stimuli [31] suggest another avenue through which raised ribosome function and specificity may be regulated.
Ribosome specialization can also be achieved through allelic variation, the most well-documented example of which comes from recent studies of rRNA. For example, the E. coli genome contains seven distinct rRNA operons, each with their specific sequence variants [32]. RNAseq analyses were used to detect differences in the utilization of specific rRNA operons in response to nutrient limitation-induced stress, and these correlated with changes in ribosome function, gene expression, and cellular physiology, thus demonstrating specific roles for rRNA allelic variants [33]. In eukaryotes, rDNA copy number varies widely, and a cursory analysis of the human and mouse rDNA sequences revealed the potential for sequence heterogeneity within rDNA operons [34]. More recently, a meta-analysis of human and mouse genome databases identified pervasive intra- and inter-individual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse, and ribosomes bearing variant rRNA alleles were found to be present in the actively translating ribosome pool [35]. These findings strengthen the idea that physically and functionally heterogeneous ribosomes may be important for normal physiological development and homeostasis and, conversely, in pathological processes. Allelic variation may also play an important role in population biology and evolution. This may provide a means though which a species could be pre-adapted to survive fluctuations in environmental conditions, e.g., climate change or location-specific differences in micronutrient availability.

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