Thank you for visiting Nature.com. The browser version you are using has limited CSS support. For the best experience, we recommend that you use an updated browser (or disable Compatibility Mode in Internet Explorer). In the meantime, to ensure continued support, we will render the site without styles and JavaScript.
The evolution of microbial parasites involves a counteraction between natural selection, which causes parasites to improve, and genetic drift, which causes parasites to lose genes and accumulate deleterious mutations. Here, in order to understand how this counteraction occurs on the scale of a single macromolecule, we describe the cryo-EM structure of the ribosome of Encephalitozoon cuniculi, a eukaryotic organism with one of the smallest genomes in nature. The extreme reduction of rRNA in E. cuniculi ribosomes is accompanied by unprecedented structural changes, such as the evolution of previously unknown fused rRNA linkers and rRNA without bulges. In addition, the E. cuniculi ribosome survived the loss of rRNA fragments and proteins by developing the ability to use small molecules as structural mimics of degraded rRNA fragments and proteins. Overall, we show that molecular structures long thought to be reduced, degenerate, and subject to debilitating mutations have a number of compensatory mechanisms that keep them active despite extreme molecular contractions.
Because most groups of microbial parasites have unique molecular tools to exploit their hosts, we often have to develop different therapeutics for different groups of parasites1,2. However, new evidence suggests that some aspects of parasite evolution are convergent and largely predictable, indicating a potential basis for broad therapeutic interventions in microbial parasites3,4,5,6,7,8,9.
Previous work has identified a common evolutionary trend in microbial parasites called genome reduction or genome decay10,11,12,13. Current research shows that when microorganisms give up their free-living lifestyle and become intracellular parasites (or endosymbionts), their genomes undergo slow but amazing metamorphoses over millions of years9,11. In a process known as genome decay, microbial parasites accumulate deleterious mutations that turn many previously important genes into pseudogenes, leading to gradual gene loss and mutational collapse14,15. This collapse can destroy up to 95% of the genes in the oldest intracellular organisms compared to closely related free-living species. Thus, the evolution of intracellular parasites is a tug-of-war between two opposing forces: Darwinian natural selection, leading to the improvement of parasites, and the collapse of the genome, throwing parasites into oblivion. How the parasite managed to emerge from this tug-of-war and retain the activity of its molecular structure remains unclear.
Although the mechanism of genome decay is not fully understood, it appears to occur mainly due to frequent genetic drift. Because parasites live in small, asexual, and genetically limited populations, they cannot effectively eliminate deleterious mutations that sometimes occur during DNA replication. This leads to irreversible accumulation of harmful mutations and reduction of the parasite genome. As a result, the parasite not only loses genes that are no longer necessary for its survival in the intracellular environment. It is the inability of parasite populations to effectively eliminate sporadic deleterious mutations that causes these mutations to accumulate throughout the genome, including their most important genes.
Much of our current understanding of genome reduction is based solely on comparisons of genome sequences, with less attention to changes in actual molecules that perform housekeeping functions and serve as potential drug targets. Comparative studies have shown that the burden of deleterious intracellular microbial mutations appears to predispose proteins and nucleic acids to misfold and aggregate, making them more chaperone dependent and hypersensitive to heat19,20,21,22,23. In addition, various parasites—independent evolution sometimes separated by as much as 2.5 billion years—experienced a similar loss of quality control centers in their protein synthesis5,6 and DNA repair mechanisms24. However, little is known about the impact of intracellular lifestyle on all other properties of cellular macromolecules, including molecular adaptation to an increasing burden of deleterious mutations.
In this work, in order to better understand the evolution of proteins and nucleic acids of intracellular microorganisms, we determined the structure of ribosomes of the intracellular parasite Encephalitozoon cuniculi. E. cuniculi is a fungus-like organism belonging to a group of parasitic microsporidia that have unusually small eukaryotic genomes and are therefore used as model organisms to study genome decay25,26,27,28,29,30. Recently, cryo-EM ribosome structure was determined for moderately reduced genomes of Microsporidia, Paranosema locustae, and Vairimorpha necatrix31,32 (~3.2 Mb genome). These structures suggest that some loss of rRNA amplification is compensated by the development of new contacts between neighboring ribosomal proteins or the acquisition of new msL131,32 ribosomal proteins. Species Encephalitozoon (genome ~2.5 million bp), along with their closest relative Ordospora, demonstrate the ultimate degree of genome reduction in eukaryotes – they have less than 2000 protein-coding genes, and it is expected that their ribosomes are not only devoid of rRNA expansion fragments (rRNA fragments that distinguish eukaryotic ribosomes from bacterial ribosomes) also have four ribosomal proteins due to their lack of homologues in the E. cuniculi genome26,27,28. Therefore, we concluded that the E. cuniculi ribosome can reveal previously unknown strategies for molecular adaptation to genome decay.
Our cryo-EM structure represents the smallest eukaryotic cytoplasmic ribosome to be characterized and provides insight into how the ultimate degree of genome reduction affects the structure, assembly, and evolution of the molecular machinery that is integral to the cell. We found that the E. cuniculi ribosome violates many of the widely conserved principles of RNA folding and ribosome assembly, and discovered a new, previously unknown ribosomal protein. Quite unexpectedly, we show that microsporidia ribosomes have evolved the ability to bind small molecules, and hypothesize that truncations in rRNA and proteins trigger evolutionary innovations that may ultimately confer useful qualities on the ribosome.
To improve our understanding of the evolution of proteins and nucleic acids in intracellular organisms, we decided to isolate E. cuniculi spores from cultures of infected mammalian cells in order to purify their ribosomes and determine the structure of these ribosomes. It is difficult to obtain a large number of parasitic microsporidia because microsporidia cannot be cultured in a nutrient medium. Instead, they grow and reproduce only inside the host cell. Therefore, to obtain E. cuniculi biomass for ribosome purification, we infected the mammalian kidney cell line RK13 with E. cuniculi spores and cultured these infected cells for several weeks to allow E. cuniculi to grow and multiply. Using an infected cell monolayer of about half a square meter, we were able to purify about 300 mg of Microsporidia spores and use them to isolate ribosomes. We then disrupted the purified spores with glass beads and isolated the crude ribosomes using stepwise polyethylene glycol fractionation of the lysates. This allowed us to obtain approximately 300 µg of raw E. cuniculi ribosomes for structural analysis.
We then collected cryo-EM images using the resulting ribosome samples and processed these images using masks corresponding to the large ribosomal subunit, small subunit head, and small subunit. During this process, we collected images of about 108,000 ribosomal particles and computed cryo-EM images with a resolution of 2.7 Å (Supplementary Figures 1-3). We then used cryoEM images to model rRNA, ribosomal protein, and hibernation factor Mdf1 associated with E. cuniculi ribosomes (Fig. 1a, b).
a Structure of the E. cuniculi ribosome in complex with the hibernation factor Mdf1 (pdb id 7QEP). b Map of hibernation factor Mdf1 associated with the E. cuniculi ribosome. c Secondary structure map comparing recovered rRNA in Microsporidian species to known ribosomal structures. The panels show the location of the amplified rRNA fragments (ES) and ribosome active sites, including the decoding site (DC), the sarcinicin loop (SRL), and the peptidyl transferase center (PTC). d The electron density corresponding to the peptidyl transferase center of the E. cuniculi ribosome suggests that this catalytic site has the same structure in the E. cuniculi parasite and its hosts, including H. sapiens. e, f The corresponding electron density of the decoding center (e) and the schematic structure of the decoding center (f) indicate that E. cuniculi has residues U1491 instead of A1491 (E. coli numbering) in many other eukaryotes. This change suggests that E. cuniculi may be sensitive to antibiotics that target this active site.
In contrast to the previously established structures of V. necatrix and P. locustae ribosomes (both structures represent the same microsporidia family Nosematidae and are very similar to each other), 31,32 E. cuniculi ribosomes undergo numerous processes of rRNA and protein fragmentation. Further denaturation (Supplementary Figures 4-6). In rRNA, the most striking changes included complete loss of the amplified 25S rRNA fragment ES12L and partial degeneration of h39, h41, and H18 helices (Fig. 1c, Supplementary Fig. 4). Among ribosomal proteins, the most striking changes included complete loss of the eS30 protein and shortening of the eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, and eS7 proteins (Supplementary Figures 4, 5).
Thus, the extreme reduction of the genomes of Encephalotozoon/Ordospora species is reflected in their ribosome structure: E. cuniculi ribosomes experience the most dramatic loss of protein content in eukaryotic cytoplasmic ribosomes subject to structural characterization, and they do not even have those rRNA and protein fragments that are widely conserved not only in eukaryotes, but also in the three domains of life. The structure of the E. cuniculi ribosome provides the first molecular model for these changes and reveals evolutionary events that have been overlooked by both comparative genomics and studies of intracellular biomolecular structure (Supplementary Fig. 7). Below, we describe each of these events along with their likely evolutionary origins and their potential impact on ribosome function.
We then found that, in addition to large rRNA truncations, E. cuniculi ribosomes have rRNA variations at one of their active sites. Although the peptidyl transferase center of the E. cuniculi ribosome has the same structure as other eukaryotic ribosomes (Fig. 1d), the decoding center differs due to sequence variation at nucleotide 1491 (E. coli numbering, Fig. 1e, f). This observation is important because the decoding site of eukaryotic ribosomes typically contains residues G1408 and A1491 compared to bacterial-type residues A1408 and G1491. This variation underlies the different sensitivity of bacterial and eukaryotic ribosomes to the aminoglycoside family of ribosomal antibiotics and other small molecules that target the decoding site. At the decoding site of the E. cuniculi ribosome, residue A1491 was replaced with U1491, potentially creating a unique binding interface for small molecules targeting this active site. The same A14901 variant is also present in other microsporidia such as P. locustae and V. necatrix, suggesting that it is widespread among microsporidia species (Fig. 1f).
Because our E. cuniculi ribosome samples were isolated from metabolically inactive spores, we tested the cryo-EM map of E. cuniculi for previously described ribosome binding under stress or starvation conditions. Hibernation factors 31,32,36,37, 38. We matched the previously established structure of the hibernating ribosome with the cryo-EM map of the E. cuniculi ribosome. For docking, S. cerevisiae ribosomes were used in complex with hibernation factor Stm138, locust ribosomes in complex with Lso232 factor, and V. necatrix ribosomes in complex with Mdf1 and Mdf231 factors. At the same time, we found the cryo-EM density corresponding to the rest factor Mdf1. Similar to Mdf1 binding to the V. necatrix ribosome, Mdf1 also binds to the E. cuniculi ribosome, where it blocks the E site of the ribosome, possibly helping to make ribosomes available when parasite spores become metabolically inactive upon body inactivation (Figure 2). ).
Mdf1 blocks the E site of the ribosome, which appears to help inactivate the ribosome when parasite spores become metabolically inactive. In the structure of the E. cuniculi ribosome, we found that Mdf1 forms a previously unknown contact with the L1 ribosome stem, the part of the ribosome that facilitates the release of deacylated tRNA from the ribosome during protein synthesis. These contacts suggest that Mdf1 dissociates from the ribosome using the same mechanism as deacetylated tRNA, providing a possible explanation for how the ribosome removes Mdf1 to reactivate protein synthesis.
However, our structure revealed an unknown contact between Mdf1 and the L1 ribosome leg (the part of the ribosome that helps release deacylated tRNA from the ribosome during protein synthesis). In particular, Mdf1 uses the same contacts as the elbow segment of the deacylated tRNA molecule (Fig. 2). This previously unknown molecular modeling showed that Mdf1 dissociates from the ribosome using the same mechanism as deacetylated tRNA, which explains how the ribosome removes this hibernation factor to reactivate protein synthesis.
When constructing the rRNA model, we found that the E. cuniculi ribosome has abnormally folded rRNA fragments, which we called fused rRNA (Fig. 3). In ribosomes that span the three domains of life, rRNA folds into structures in which most rRNA bases either base pair and fold with each other or interact with ribosomal proteins38,39,40. However, in E. cuniculi ribosomes, rRNAs seem to violate this folding principle by converting some of their helices into unfolded rRNA regions.
Structure of the H18 25S rRNA helix in S. cerevisiae, V. necatrix, and E. cuniculi. Typically, in ribosomes spanning the three life domains, this linker coils into an RNA helix that contains 24 to 34 residues. In Microsporidia, in contrast, this rRNA linker is gradually reduced to two single-stranded uridine-rich linkers containing only 12 residues. Most of these residues are exposed to solvents. The figure shows that parasitic microsporidia appear to violate the general principles of rRNA folding, where rRNA bases are usually coupled to other bases or involved in rRNA-protein interactions. In microsporidia, some rRNA fragments take on an unfavorable fold, in which the former rRNA helix becomes a single-stranded fragment elongated almost in a straight line. The presence of these unusual regions allows microsporidia rRNA to bind distant rRNA fragments using a minimal number of RNA bases.
The most striking example of this evolutionary transition can be observed in the H18 25S rRNA helix (Fig. 3). In species from E. coli to humans, the bases of this rRNA helix contain 24-32 nucleotides, forming a slightly irregular helix. In previously identified ribosomal structures from V. necatrix and P. locustae,31,32 the bases of the H18 helix are partially uncoiled, but nucleotide base pairing is preserved. However, in E. cuniculi this rRNA fragment becomes the shortest linkers 228UUUGU232 and 301UUUUUUUUU307. Unlike typical rRNA fragments, these uridine-rich linkers do not coil or make extensive contact with ribosomal proteins. Instead, they adopt solvent-open and fully unfolded structures in which the rRNA strands are extended almost straight. This stretched conformation explains how E. cuniculi uses only 12 RNA bases to fill the 33 Å gap between the H16 and H18 rRNA helices, while other species require at least twice as many rRNA bases to fill the gap.
Thus, we can demonstrate that, through energetically unfavorable folding, parasitic microsporidia have developed a strategy to contract even those rRNA segments that remain broadly conserved across species in the three domains of life. Apparently, by accumulating mutations that transform rRNA helices into short poly-U linkers, E. cuniculi can form unusual rRNA fragments containing as few nucleotides as possible for ligation of distal rRNA fragments. This helps explain how microsporidia achieved a dramatic reduction in their basic molecular structure without losing their structural and functional integrity.
Another unusual feature of E. cuniculi rRNA is the appearance of rRNA without thickenings (Fig. 4). Bulges are nucleotides without base pairs that twist out of the RNA helix instead of hiding in it. Most rRNA protrusions act as molecular adhesives, helping to bind adjacent ribosomal proteins or other rRNA fragments. Some of the bulges act as hinges, allowing the rRNA helix to flex and fold optimally for productive protein synthesis 41 .
a An rRNA protrusion (S. cerevisiae numbering) is absent from the E. cuniculi ribosome structure, but present in most other eukaryotes b E. coli, S. cerevisiae, H. sapiens, and E. cuniculi internal ribosomes. parasites lack many of the ancient, highly conserved rRNA bulges. These thickenings stabilize the ribosome structure; therefore, their absence in microsporidia indicates a reduced stability of rRNA folding in microsporidia parasites. Comparison with P stems (L7/L12 stems in bacteria) shows that the loss of rRNA bumps sometimes coincides with the appearance of new bumps next to the lost bumps. The H42 helix in the 23S/28S rRNA has an ancient bulge (U1206 in Saccharomyces cerevisiae) estimated to be at least 3.5 billion years old due to its protection in three domains of life. In microsporidia, this bulge is eliminated. However, a new bulge appeared next to the lost bulge (A1306 in E. cuniculi).
Strikingly, we found that E. cuniculi ribosomes lack most of the rRNA bulges found in other species, including more than 30 bulges conserved in other eukaryotes (Fig. 4a). This loss eliminates many contacts between ribosomal subunits and adjacent rRNA helices, sometimes creating large hollow voids within the ribosome, making the E. cuniculi ribosome more porous compared to more traditional ribosomes (Fig. 4b). Notably, we found that most of these bulges were also lost in the previously identified V. necatrix and P. locustae ribosome structures, which were overlooked by previous structural analyzes31,32.
Sometimes the loss of rRNA bulges is accompanied by the development of new bulges next to the lost bulge. For example, the ribosomal P-stem contains a U1208 bulge (in Saccharomyces cerevisiae) that survived from E. coli to humans and is therefore estimated to be 3.5 billion years old. During protein synthesis, this bulge helps the P stem move between open and closed conformations so that the ribosome can recruit translation factors and deliver them to the active site. In E. cuniculi ribosomes, this thickening is absent; however, a new thickening (G883) located only in three base pairs can contribute to the restoration of the optimal flexibility of the P stem (Fig. 4c).
Our data on rRNA without bulges suggest that rRNA minimization is not limited to the loss of rRNA elements on the surface of the ribosome, but may also involve the ribosome nucleus, creating a parasite-specific molecular defect that has not been described in free-living cells. living species are observed.
After modeling canonical ribosomal proteins and rRNA, we found that conventional ribosomal components cannot explain the three parts of the cryo-EM image. Two of these fragments are small molecules in size (Fig. 5, Supplementary Fig. 8). The first segment is sandwiched between the ribosomal proteins uL15 and eL18 in a position usually occupied by the C-terminus of eL18, which is shortened in E. cuniculi. Although we cannot determine the identity of this molecule, the size and shape of this density island is well explained by the presence of spermidine molecules. Its binding to the ribosome is stabilized by microsporidia-specific mutations in the uL15 proteins (Asp51 and Arg56), which seem to increase the affinity of the ribosome for this small molecule, as they allow uL15 to wrap the small molecule into a ribosomal structure. Supplementary Figure 2). 8, additional data 1, 2).
Cryo-EM imaging showing the presence of nucleotides outside the ribose bound to the E. cuniculi ribosome. In the E. cuniculi ribosome, this nucleotide occupies the same place as the 25S rRNA A3186 nucleotide (Saccharomyces cerevisiae numbering) in most other eukaryotic ribosomes. b In the ribosomal structure of E. cuniculi, this nucleotide is located between the ribosomal proteins uL9 and eL20, thereby stabilizing the contact between the two proteins. cd eL20 sequence conservation analysis among microsporidia species. The phylogenetic tree of Microsporidia species (c) and multiple sequence alignment of the eL20 protein (d) show that nucleotide-binding residues F170 and K172 are conserved in most typical Microsporidia, with the exception of S. lophii, with the exception of early branching Microsporidia, which retained the ES39L rRNA extension. e This figure shows that nucleotide-binding residues F170 and K172 are present only in eL20 of the highly reduced microsporidia genome, but not in other eukaryotes. Overall, these data suggest that Microsporidian ribosomes have developed a nucleotide binding site that appears to bind AMP molecules and use them to stabilize protein-protein interactions in the ribosomal structure. The high conservation of this binding site in Microsporidia and its absence in other eukaryotes suggests that this site may provide a selective survival advantage for Microsporidia. Thus, the nucleotide-binding pocket in the microsporidia ribosome does not appear to be a degenerate feature or end form of rRNA degradation as previously described, but rather a useful evolutionary innovation that allows the microsporidia ribosome to directly bind small molecules, using them as molecular building blocks. building blocks for ribosomes. This discovery makes the microsporidia ribosome the only ribosome known to use a single nucleotide as its structural building block. f Hypothetical evolutionary pathway derived from nucleotide binding.
The second low molecular weight density is located at the interface between ribosomal proteins uL9 and eL30 (Fig. 5a). This interface was previously described in the structure of the Saccharomyces cerevisiae ribosome as a binding site for the 25S nucleotide of rRNA A3186 (part of the ES39L rRNA extension)38. It was shown that in degenerate P. locustae ES39L ribosomes, this interface binds an unknown single nucleotide 31, and it is assumed that this nucleotide is a reduced final form of rRNA, in which the length of rRNA is ~130-230 bases. ES39L is reduced to a single nucleotide 32.43. Our cryo-EM images support the idea that density can be explained by nucleotides. However, the higher resolution of our structure showed that this nucleotide is an extraribosomal molecule, possibly AMP (Fig. 5a, b).
We then asked whether the nucleotide binding site appeared in the E. cuniculi ribosome or whether it existed previously. Since nucleotide binding is mainly mediated by the Phe170 and Lys172 residues in the eL30 ribosomal protein, we assessed the conservation of these residues in 4396 representative eukaryotes. As in the case of uL15 above, we found that the Phe170 and Lys172 residues are highly conserved only in typical Microsporidia, but absent in other eukaryotes, including atypical Microsporidia Mitosporidium and Amphiamblys, in which the ES39L rRNA fragment is not reduced 44, 45, 46 (Fig. 5c). -e).
Taken together, these data support the idea that E. cuniculi and possibly other canonical microsporidia have evolved the ability to efficiently capture large numbers of small metabolites in the ribosome structure to compensate for the decline in rRNA and protein levels. In doing so, they have developed a unique ability to bind nucleotides outside the ribosome, showing that parasitic molecular structures compensate by capturing abundant small metabolites and using them as structural mimics of degraded RNA and protein fragments. .
The third unsimulated part of our cryo-EM map, found in the large ribosomal subunit. The relatively high resolution (2.6 Å) of our map suggests that this density belongs to proteins with unique combinations of large side chain residues, which allowed us to identify this density as a previously unknown ribosomal protein that we identified as It was named msL2 (Microsporidia- specific protein L2) (methods, figure 6). Our homology search showed that msL2 is conserved in the Microsporidia clade of the genus Encephaliter and Orosporidium, but absent in other species, including other Microsporidia. In the ribosomal structure, msL2 occupies a gap formed by the loss of the extended ES31L rRNA. In this void, msL2 helps stabilize rRNA folding and can compensate for the loss of ES31L (Figure 6).
a Electron density and model of the Microsporidia-specific ribosomal protein msL2 found in E. cuniculi ribosomes. b Most eukaryotic ribosomes, including the 80S ribosome of Saccharomyces cerevisiae, have ES19L rRNA amplification lost in most Microsporidian species. The previously established structure of the V. necatrix microsporidia ribosome suggests that the loss of ES19L in these parasites is compensated by the evolution of the new msL1 ribosomal protein. In this study, we found that the E. cuniculi ribosome also developed an additional ribosomal RNA mimic protein as an apparent compensation for the loss of ES19L. However, msL2 (currently annotated as the hypothetical ECU06_1135 protein) and msL1 have different structural and evolutionary origins. c This discovery of the generation of evolutionarily unrelated msL1 and msL2 ribosomal proteins suggests that if ribosomes accumulate detrimental mutations in their rRNA, they can achieve unprecedented levels of compositional diversity in even a small subset of closely related species. This discovery could help clarify the origin and evolution of the mitochondrial ribosome, which is known for its highly reduced rRNA and abnormal variability in protein composition across species.
We then compared the msL2 protein with the previously described msL1 protein, the only known microsporidia-specific ribosomal protein found in the V. necatrix ribosome. We wanted to test whether msL1 and msL2 are evolutionarily related. Our analysis showed that msL1 and msL2 occupy the same cavity in the ribosomal structure, but have different primary and tertiary structures, which indicates their independent evolutionary origin (Fig. 6). Thus, our discovery of msL2 provides evidence that groups of compact eukaryotic species can independently evolve structurally distinct ribosomal proteins to compensate for the loss of rRNA fragments. This finding is notable in that most cytoplasmic eukaryotic ribosomes contain an invariant protein, including the same family of 81 ribosomal proteins. The appearance of msL1 and msL2 in various clades of microsporidia in response to the loss of extended rRNA segments suggests that degradation of the parasite’s molecular architecture causes parasites to seek compensatory mutations, which may eventually lead to their acquisition in different parasite populations. structures.
Finally, when our model was completed, we compared the composition of the E. cuniculi ribosome with that predicted from the genome sequence. Several ribosomal proteins, including eL14, eL38, eL41, and eS30, were previously thought to be missing from the E. cuniculi genome due to the apparent absence of their homologues from the E. cuniculi genome. Loss of many ribosomal proteins is also predicted in most other highly reduced intracellular parasites and endosymbionts. For example, although most free-living bacteria contain the same family of 54 ribosomal proteins, only 11 of these protein families have detectable homologues in each analyzed genome of host-restricted bacteria. In support of this notion, a loss of ribosomal proteins has been experimentally observed in V. necatrix and P. locustae microsporidia, which lack the eL38 and eL4131,32 proteins.
However, our structures show that only eL38, eL41, and eS30 are actually lost in the E. cuniculi ribosome. The eL14 protein was conserved and our structure showed why this protein could not be found in the homology search (Fig. 7). In E. cuniculi ribosomes, most of the eL14 binding site is lost due to degradation of the rRNA-amplified ES39L. In the absence of ES39L, eL14 lost most of its secondary structure, and only 18% of the eL14 sequence was identical in E. cuniculi and S. cerevisiae. This poor sequence preservation is remarkable because even Saccharomyces cerevisiae and Homo sapiens—organisms that evolved 1.5 billion years apart—share more than 51% of the same residues in eL14. This anomalous loss of conservation explains why E. cuniculi eL14 is currently annotated as the putative M970_061160 protein and not as the eL1427 ribosomal protein.
and The Microsporidia ribosome lost the ES39L rRNA extension, which partially eliminated the eL14 ribosomal protein binding site. In the absence of ES39L, the eL14 microspore protein undergoes a loss of secondary structure, in which the former rRNA-binding α-helix degenerates into a minimal length loop. b Multiple sequence alignment shows that the eL14 protein is highly conserved in eukaryotic species (57% sequence identity between yeast and human homologues), but poorly conserved and divergent in microsporidia (in which no more than 24% of residues are identical to the eL14 homologue). from S. cerevisiae or H. sapiens). This poor sequence conservation and secondary structure variability explains why the eL14 homologue has never been found in E. cuniculi and why this protein is thought to have been lost in E. cuniculi. In contrast, E. cuniculi eL14 was previously annotated as a putative M970_061160 protein. This observation suggests that microsporidia genome diversity is currently overestimated: some genes currently thought to be lost in microsporidia are in fact preserved, albeit in highly differentiated forms; instead, some are thought to code for microsporidia genes for worm-specific proteins (e.g., the hypothetical protein M970_061160) actually codes for the very diverse proteins found in other eukaryotes.
This finding suggests that rRNA denaturation can lead to a dramatic loss of sequence conservation in adjacent ribosomal proteins, rendering these proteins undetectable for homology searches. Thus, we may overestimate the actual degree of molecular degradation in small genome organisms, since some proteins thought to be lost actually persist, albeit in highly altered forms.
How can parasites retain the function of their molecular machines under conditions of extreme genome reduction? Our study answers this question by describing the complex molecular structure (ribosome) of E. cuniculi, an organism with one of the smallest eukaryotic genomes.
It has been known for almost two decades that protein and RNA molecules in microbial parasites often differ from their homologous molecules in free-living species because they lack quality control centers, are reduced to 50% of their size in free-living microbes, etc. . many debilitating mutations that impair folding and function. For example, the ribosomes of small genome organisms, including many intracellular parasites and endosymbionts, are expected to lack several ribosomal proteins and up to one third of rRNA nucleotides compared to free-living species 27, 29, 30, 49. However, the way these molecules function in parasite remains largely a mystery, studied mainly through comparative genomics.
Our study shows that the structure of macromolecules can reveal many aspects of evolution that are difficult to extract from traditional comparative genomic studies of intracellular parasites and other host-restricted organisms (Supplementary Fig. 7). For example, the example of the eL14 protein shows that we can overestimate the actual degree of degradation of the molecular apparatus in parasitic species. Encephalitic parasites are now believed to have hundreds of microsporidia-specific genes. However, our results show that some of these seemingly specific genes are actually just very different variants of genes that are common in other eukaryotes. Moreover, the example of the msL2 protein shows how we overlook new ribosomal proteins and underestimate the content of parasitic molecular machines. The example of small molecules shows how we can overlook the most ingenious innovations in parasitic molecular structures that can give them new biological activity.
Taken together, these results improve our understanding of the differences between the molecular structures of host-restricted organisms and their counterparts in free-living organisms. We show that molecular machines, long thought to be reduced, degenerate, and subject to various debilitating mutations, instead have a set of systematically overlooked unusual structural features.
On the other hand, the non-bulky rRNA fragments and fused fragments that we found in the ribosomes of E. cuniculi suggest that genome reduction can change even those parts of the basic molecular machinery that are preserved in the three domains of life – after almost 3.5 billion years . independent evolution of species.
The bulge-free and fused rRNA fragments in E. cuniculi ribosomes are of particular interest in the light of previous studies of RNA molecules in endosymbiotic bacteria. For example, in the aphid endosymbiont Buchnera aphidicola, rRNA and tRNA molecules have been shown to have temperature-sensitive structures due to A+T composition bias and a high proportion of non-canonical base pairs20,50. These changes in RNA, as well as changes in protein molecules, are now thought to be responsible for the overdependence of endosymbionts on partners and the inability of endosymbionts to transfer heat 21, 23 . Although parasitic microsporidia rRNA has structurally distinct changes, the nature of these changes suggests that reduced thermal stability and higher dependence on chaperone proteins may be common features of RNA molecules in organisms with reduced genomes.
On the other hand, our structures show that parasite microsporidia have evolved a unique ability to resist broadly conserved rRNA and protein fragments, developing the ability to use abundant and readily available small metabolites as structural mimics of degenerate rRNA and protein fragments. Molecular structure degradation. . This opinion is supported by the fact that small molecules that compensate for the loss of protein fragments in the rRNA and ribosomes of E. cuniculi bind to microsporidia-specific residues in the uL15 and eL30 proteins. This suggests that binding of small molecules to ribosomes may be a product of positive selection, in which Microsporidia-specific mutations in ribosomal proteins have been selected for their ability to increase the affinity of ribosomes for small molecules, which may lead to more efficient ribosomal organisms. The discovery reveals a smart innovation in the molecular structure of microbial parasites and gives us a better understanding of how parasite molecular structures maintain their function despite reductive evolution.
At present, the identification of these small molecules remains unclear. It is not clear why the appearance of these small molecules in the ribosomal structure differs between microsporidia species. In particular, it is not clear why nucleotide binding is observed in the ribosomes of E. cuniculi and P. locustae, and not in the ribosomes of V. necatrix, despite the presence of the F170 residue in the eL20 and K172 proteins of V. necatrix. This deletion may be caused by residue 43 uL6 (located adjacent to the nucleotide binding pocket), which is tyrosine in V. necatrix and not threonine in E. cuniculi and P. locustae. The bulky aromatic side chain of Tyr43 can interfere with nucleotide binding due to steric overlap. Alternatively, the apparent nucleotide deletion may be due to the low resolution of cryo-EM imaging, which hinders the modeling of V. necatrix ribosomal fragments.
On the other hand, our work suggests that the process of genome decay may be an inventive force. In particular, the structure of the E. cuniculi ribosome suggests that the loss of rRNA and protein fragments in the microsporidia ribosome creates evolutionary pressure that promotes changes in ribosome structure. These variants occur far from the active site of the ribosome and appear to help maintain (or restore) optimal ribosome assembly that would otherwise be disrupted by reduced rRNA. This suggests that a major innovation of the microsporidia ribosome appears to have evolved into a need to buffer gene drift.
Perhaps this is best illustrated by nucleotide binding, which has never been observed in other organisms so far. The fact that nucleotide-binding residues are present in typical microsporidia, but not in other eukaryotes, suggests that nucleotide-binding sites are not just relics waiting to disappear, or the final site for rRNA to be restored to the form of individual nucleotides. Instead, this site seems like a useful feature that could have evolved over several rounds of positive selection. Nucleotide binding sites may be a by-product of natural selection: once ES39L is degraded, microsporidia are forced to seek compensation to restore optimal ribosome biogenesis in the absence of ES39L. Since this nucleotide can mimic the molecular contacts of the A3186 nucleotide in ES39L, the nucleotide molecule becomes a building block of the ribosome, the binding of which is further improved by mutation of the eL30 sequence.
With regard to the molecular evolution of intracellular parasites, our study shows that the forces of Darwinian natural selection and genetic drift of genome decay do not operate in parallel, but oscillate. First, genetic drift eliminates important features of biomolecules, making compensation sorely needed. Only when parasites satisfy this need through Darwinian natural selection will their macromolecules have a chance to develop their most impressive and innovative traits. Importantly, the evolution of nucleotide binding sites in the E. cuniculi ribosome suggests that this loss-to-gain pattern of molecular evolution not only amortizes deleterious mutations, but sometimes confers entirely new functions on parasitic macromolecules.
This idea is consistent with Sewell Wright’s moving equilibrium theory, which states that a strict system of natural selection limits the ability of organisms to innovate51,52,53. However, if genetic drift disrupts natural selection, these drifts can produce changes that are not in themselves adaptive (or even detrimental) but lead to further changes that provide higher fitness or new biological activity. Our framework supports this idea by illustrating that the same type of mutation that reduces the fold and function of a biomolecule appears to be the main trigger for its improvement. In line with the win-win evolutionary model, our study shows that genome decay, traditionally viewed as a degenerative process, is also a major driver of innovation, sometimes and perhaps even often allowing macromolecules to acquire new parasitic activities. can use them.