Structural analysis of mitochondrial rRNA gene variants identified in patients with deafness

The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants.


Table of Contents
The 74U>C(721U>C) base change has been recurrently found associated with hearing impairment (Guaran et al., 2013, Elstner et al., 2008, 2004, Rydzanicz et al., 2010, Konings et al., 2008. This base change is moderately abundant with 167 GenBank appearances to date (Ruiz-Pesini et al., 2007). Position 74U (721U) is unpaired and stacks onto position 105C>U (m.752C>U), where a C>U base change has been found in a patient with sensorineural hearing loss (Li et al., 2005). The fact that neither position uses its base to make contacts to other mito-ribosomal residues argues against their potential pathogenicity.
-88A>G (m.735A>G): This variant was detected by Mkaouar-Rebai et al. in two unrelated probands with sensorineural hearing loss (Mkaouar-Rebai et al., 2010). In the secondary structure map of 12S mt-rRNA, position 88A (m.735A) lies in the capping loop of h7. The loop was not resolved in the original structure of the human ribosome (3J9M) but could be modeled in the 2.2-Å structure of the mito-ribosome, albeit at a lower resolution, likely indicating high flexibility in this region. Despite the lack of resolution in the region, the unpaired 88A (m.735A) has been modeled sandwiched between Arg 68 of MRPS26/mS26 (pink in the structure) and the adjacent 89C (m.736C) is also in the neighborhood of the closing loop of h7 but far from the base of 88A (m.735A). All these observations, together with the lack of hydrogen bonding interactions involving the base of 88A (m.735A), strongly suggest that the G>A base change at 88A (m.735A) is phenotypically silent.
-154A>G (m.801 A>G): The 154A>G (m.801A>G) highly rare variant was found by Lu et al. (Lu et al., 2010) in a pediatric patient with hearing loss. Position 154A (m.801A) forms a non-canonical A•A base pair with 67A (m.714A) at the junction between h6a and h14 (not shown). The base pair, together with the preceding triple base interaction involving the 153C:68G (m.800C:m.715G) base pair in h6a and position 169A (m.816A), located in the rRNA segment linking h14 and h5, likely aids in maintaining the higher order structure at the junction of these three rRNA helices. The A•A base pair is replaced in S. scrofa by a G•A mismatch (RCSB ID: 5AJ4), suggesting the existence of enough flexibility in this helical region to tolerate slight deviations in the relative orientation of the bases forming this base pair. This view would be in agreement with the idea that the 154A>G (m.801A>G) replacement, resulting in an A•G mismatch, could be easily tolerated.

166A>G (m.813A>G):
Identified in two studies (Guaran et al., 2013, Rydzanicz et al., 2010. Adjacent to bridge B5, which involves residues 164 (m.811) and 165A (m.812A) in 12S mt-rRNA and MRPL14/uL14m in the LSU (Amunts et al., 2015). Position 166A (m.813A), stacks onto the adjacent base of 166A (m.813A) and is too far to interact with other mito-ribosomal residues or ligands. Despite its location near a functionally important mito-ribosomal element, there is no evidence that the base change might induce a structural clash.
-192A>G (m.839A>G): The 192A>G (m.839A>G) variant was found in a patient with non-syndromic hearing loss (Lu et al., 2010).  (Guaran et al., 2013, Elstner et al., 2008, Rydzanicz et al., 2010, Li et al., 2005, Lu et al., 2010, Human et al., 2010, Yano et al., 2014, Rydzanicz et al., 2009, Mkaouar-Rebai et al., 2008, Igumnova et al., 2019. The fact that all these residues map to the back of the subunit, strongly suggests a lack of involvement in mito-ribosomal function. In agreement with this idea, the lack of density surrounding all of these positions, except for 309C (m.956C) and 394A (m.1041A), indicates that the structure of this RNA segment is highly disordered and flexible. Hence, according to the available structural data, there is no reason to think that any of the base changes reported at positions 312C (m.959C), 313C (m.960C), 314U (m.961U), and 315C (m.962C) could not be silently accommodated in the structure. Indeed, two of the reported variants, namely 313Ins (m.960ins) and 314U>G (m.961U>G) are known haplotype markers. The 312C>U (m.959C>U) variant was found together with the LSU 49G>A (m.1719G>A) base change and the 35delG allele of the connexin26 gene GJB2 in heteroplasmy (Guaran et al., 2013). Since both 312C>U (m.959C>U) and the LSU variant 49G>A (m.1719G>A) are likely polymorphisms (see below), the possibility that the GJB2 35delG mutation may be the only cause of deafness in this patient appears as the most probable explanation for the observed symptoms (Guaran et al., 2013, Estivill et al., 1998. Even the variants identified at the two ordered bases residues above, namely 309C (m.956C) and 394A (m.1041A) are expected to be polymorphisms. Position 309C (m.956C) (Konings et al., 2008) is part of a stretch of five consecutive Cs, only two of which are clearly ordered. As for the 394A>G (m.1041A>G) variant (Konings et al., 2008), lying on the RNA strand opposite to the run of residues 309-15 (m.956-62), its base is stacked onto the guanidinium group of Arg 123 of MRPS15/uS15. Without any other involvement in structural stabilization, an A>G base change should, in principle, be well tolerated at this position. Despite all the presented evidence, the concentration of putatively pathogenic mutations in this region of 12S mt-rRNA is intriguing and perhaps reflects the existence of low selective pressure against mutations in this region.
-371G>A (m.1018G>A): Two variants map to the neighborhood of helix h23a. The haplotype marker 371G>A (m.1018G>A) variant was identified by several authors in a hearing impaired patient (Elstner et al., 2008, Konings et al., 2008. In the secondary structure map of 12S mt-rRNA, 371G (m.1018G) maps to the single-stranded stretch joining h23 and h23a. The base of 371G (m.1018G), together with the adjacent residues (positions 369-70 (m.1016-7)), is involved in a network of water-mediated hydrogen bonds that stabilizes the highly contorted conformation of the single-stranded stretch. This unusual conformation creates sites of recognition for proteins MRPS18C/bS18c (orange in structure), MRPS21/bS21 (light green in structure), and MRPS37/mS37 (color cornflower blue in structure). The equivalent position in the S. scrofa mito-ribosome is an A (RCSB ID: 5AJ4), strongly suggesting that the G>A variant at 371 (m.1018) is silent.
-500G>A (m.1147G>A): The 500G>A (m.1147G>A) variant was found in an Iranian individual with hearing loss and directly submitted to MITOMAP (Farhadi 2016, Ruiz-Pesini et al., 2007. Position 500G (m.1147G) forms a G•U wobble with position 511U (m.1158U), located at the distal helical portion of h27. A hydrogen bond between MRPS38/mS38 Arg 161 (orange in the structure) and the RNA backbone at the adjacent 499C (m.1146C) is observed. Protein MRPS38/mS38 is part of the functionally important bridge mB4 (see below). The 500G>A (m.1147G>A) variant would result in the substitution of a G•U wobble with a Watson-Crick A:U at the end of a helical segment, which in principle should be well tolerated (Ananth et al., 2013). Support for this idea comes from the fact that even a geometrically divergent U •U base pair with two hydrogen bonds is present in the S. scrofa mito-ribosome in place of the human Watson: Crick configuration of the 500G:511U (m.1147G:1158U) base pair (Greber et al., 2015). Hence, the 500G>A (m.1147G>A) variant is regarded as silent, in agreement with a previous report (Haumann et al., 2020).
The 507A>C (m.1154A>C) variant was identified in the 12S rRNA of a patient with paternally inherited hearing loss (Konings et al., 2008) and analyzed by HIA by our group before the arrival of high-resolution structures of the mammalian mito-ribosome (Smith et al., 2014). The E. coli equivalent to 507A (m.1154A) is A901 (Smith et al., 2014). Position 507A (m.1154A) is located in intersubunit bridge mB3, involving helices h27 and H67 of 12S and 16S mt-rRNA, respectively (Amunts et al., 2015). Bridge mB3 is the pivot of inter-subunit movement and remains intact during rotation (Amunts et al., 2015). Two clear hydrogen bonds are observed from Arg 144 and Arg 155 of MRPS38/mS38 (orange in the structure) to the RNA backbone at position 507A (m.1154A), whereas the base of this residue is stacked under Arg 151. Protein MRPS38/mS38 is part of the neighboring bridge mB4, a mitochondrial-specific bridge that buries the largest amount of surface area of all mito-ribosomal bridges (Amunts et al., 2015).
Perhaps the presence of MRPS38/mS38 in the mitochondrial ribosome explains the structural differences between the bacterial and mitochondrial distal end of h27. For example, the bacterial heterologous equivalent to 507A (m.1154A), position A901, is involved in tertiary contacts within helix b-h27 (Smith et al., 2014). No such contacts involving the base of 507A (m.1154A) are observed in the human mito-ribosome (Khawaja et al., 2020, Itoh et al., 2021, Itoh et al., 2022. In light of this evidence, the bacterial mutagenesis data used to support the "likely disruptive" assignment for this mutation in our previous report has to be reconsidered (Smith et al., 2014). In particular, the fact that the S. scrofa mito-ribosome possesses a U at the equivalent position (505U in structure 5AJ4), clearly supports the idea that the 507A>C (m.1154A>C) variant is likely silent, in agreement with the paternal inheritance of hearing impairment in this subject. The base C>U base change at position 758C (m.1405C) was identified together with 35delG/35delG in GJB2 (Guaran et al., 2013). The 759U>C (m.1406U>C) variant was identified in association to deafness in three studies (Guaran et al., 2013, Konings et al., 2008, Rydzanicz et al., 2010. Both 758C (m.1405C) and 759U (m.1406U) are unpaired in the loop capping h42. No hydrogen bonds involving either base are observed, strongly suggesting that both base changes can be tolerated with no effect.
-773U>C (m.1420 U>C): The variant 773U>C (m.1420 U>C) was identified by Li et al. in a Chinese pediatric subject with aminoglycoside-induced and non-syndromic hearing loss (Li et al., 2005). Position 773U (m.1420 U) is an unpaired residue located on the single-stranded strand connecting helices h29 and h42. Positions 775G (m.1422G) and 776A (m.1423A) establish Type II and Type I minor interactions, respectively, with P-site tRNA (Lancaster and Noller., 2005). Not surprisingly, almost all mutations at the bacterial equivalents of these positions, the nearly universally conserved G1338 and A1339 blocked translation (Abdi and Fredrick., 2005). The fact that the base of 773U (m.1420 U) is too far from all neighboring residues to establish hydrogen bonds and that an A and a C are the heterologous equivalents of this residue in the S. scrofa mito-ribosome and the E. coli ribosome, respectively strongly suggest that the 773U>C (m.1420 U>C) variant is silent.
-870A>C (m.1517A>C) 883A>G (m.1530A>G): Two variants map to the distal half of helix h44, specifically at positions 870A>C (m.1517A>C) and 883A>G (m.1530A>G) (Konings et al., 2008, 1766Mkaouar-Rebai,E. 2008). Instead of a helical element, this region of h44 has been modeled as two stretches of unpaired RNA running in an antiparallel fashion. Both 870A (m.1517A) and 883A (m.1530A) are unpaired and with their bases not involved in any hydrogen bond, strongly arguing for the consideration of the two variants as silent. The 30U>C (m.1700U>C) haplotype marker was found in association to deafness by Guaran et al. (Guaran et al., 2013). In the secondary structure map of 16S mt-rRNA, 30U (m.1700U) maps to the long single-stranded region connecting helices H4 and H11. Two hydrogen bonds are visible from Arg 196 of MRPL47/uL29 to the RNA backbone at position 30U (m.1700U). However, the base of 30U (m.1700U) does not participate in any hydrogen bonds. Replaced by a C at the equivalent position in the S. scrofa mito-ribosome (position 32 in RCSB ID: 7NSH). In the pig structure 32C establishes two hydrogen bonds with adenine 36A (equivalent to 34U (m.1704U) in the human mito-ribosome). Consistent with a polymorphism.
Guaran et al. found a second variant in the same single-stranded stretch, namely 38A>U (m.1708A>U) (Guaran et al., 2013). This residue establishes two hydrogen bonds with protein MRPL37/mL37, one involving the base via its amino group and the other involving the ribose. In the S. scrofa mito-ribosome, 38A (m.1708A) is replaced with a C (position 40 in RCSB ID: 7NSH), which also participates in a hydrogen bond with MRPL37/mL37. Given the distal location of the 38A>U (m.1708A>U) variant, away from all functional centers, its pathogenic character remains unclear.
The 49G>A (m.1719G>A) variant, mapping to the end of the single-stranded stretch connecting helices h4 and h11 was also found by Guaran et al. (Guaran et al., 2013), together with the 12S mt-rRNA C>U variant at position 312 (m.959) and the 35delG allele of GJB2 in heteroplasmy. Residue 49G (m.1719G) makes a sheared base pair with 339G (m.2009G). Several hydrogen bonds are observed from protein MRPL51/mL43 to the RNA backbone at positions 339-40 (m.2009-10). A hydrogen bond from is also formed between Arg 75 of MRPL9 to the RNA backbone at position 48A (m.1718A). Position 49G (m.1719G) is replaced by an A at position 51 of 16S mt-rRNA in the S. scrofa mito-ribosome, without disrupting the sheared base pair, hence arguing for the fact that 49G>A (m.1719G>A) is a polymorphism. As mentioned above for the 312C>U (m.959C>U) variant, the 35delG allele in GJB2 (or other unknown factor/s) is the most probable cause for the deafness observed in this patient (Guaran et al., 2013).

-(m.1811A>G) 141A>G:
The haplotype marker (m.1811A>G) 141A>G was identified in association with deafness by Guaran et al. (Guaran et al., 2013). The base near the back of the subunit and not involved in any hydrogen bonding interaction, underscoring its unimportant role in mito-ribosomal structure.

-399U>C (m.2069U>C):
The 399U>C (m.2069U>C) variant was found in a Japanese patient with hearing loss (Yano et al., 2014). In the 2.2-Å structure of the human mito-ribosome, position 399U (m.2069U) was modeled with its base pointing away from any potential hydrogen bond interaction, despite the lack of clear density around it. Nevertheless, this configuration is consistent with that observed in the structure of the S. scrofa mitochondrial LSU for its heterologous equivalent residue, namely position 404C (RCBS ID: 7NSH) (Kummer et al., 2021).

-568A>G (m.2238A>G):
The variant 568A>G (m.2238A>G) was found together with the SSU 448U>C (m.1095U>C) variant (Zhao et al., 2004b). In the secondary structure map of 16S mt-rRNA, position 568A (m.2238A) is located in the distal single-stranded stretch connecting H41 to H43 and H44. Within this unpaired stretch, the base of 568A (m.2238A) does not play any particular structural role besides stacking within the single RNA strand. Hence, the A>G base change at this position is not expected to have any structural effects. MRPL15/uL15 is also very close but it does not establish direct contacts with the loop. All of this suggests that the structure of this loop is important for the recognition of these proteins during assembly. In agreement with this, it has been shown that missense mutations in MRPL44 seriously affect the assembly of the large ribosomal subunit and the stability of 16S rRNA, resulting in the lack of complex IV (Wang et al., 2021, Carroll et al., 2013, Distelmaier et al., 2015. This protein:RNA arrangement is conserved in the mammalian mito-ribosome but completely absent in its bacterial counterpart. In S. scrofa (het. Equiv. 617C in 5AJ4 or 619C in the newer structures, 7NSH) the U•C base pair has been replaced by a C•U with the same geometry (density supported in 7NSH, 3.20-Å resolution). Taking all this evidence together, a picture emerges in which the loop atop H46 functions as a hub for the binding of a number of mito-ribosomal proteins during the assembly of the subunit. In this scenario, the 615U>C/G (m.2285U>C/G) variants could introduce enough distortion in the structure of the loop to cause assembly defects.
-964U>C (m.2634U>C): The rare variant 964U>C (m.2634U>C) was found in a patient with non-syndromic hearing loss (Yano et al., 2014). Position 964U (m.2634U) is located to the RNA stretch linking H67 and H68 where it positions its base away from any potential hydrogen-bonding interaction. A U is also found at the heterologous equivalent position in the S. scrofa mito-ribosome, the human and plasmodium falciparum 80S, and in the bacterial ribosome. In some of these structures, the base is within hydrogen bonding distance to the backbone of H68. However, the equivalent distances in the human mito-ribosome are too long for such an interaction.

-1543A>G (m.3213A>G):
The 1543A>G (m.3213A>G) variant was identified by Guaran et al. together with A3348G, G3591A, A3714G, G7642A, and G7805A (Guaran et al., 2013). Position 1543A (m.3213A) base pairs with 1557U (m.3227U) in H100, adjacent to the 3'end of 16S mt-rRNA. Position 1540C (m.3210C) establishes a single hydrogen bond with 1557U (m.3227U), thus creating a triple base interaction. Numerous proteins contact H100. Several MRPL3/uL3m residues contact the RNA backbone of H100 at positions (m.3228U). Additionally, MRPL3/uL3m Arg 156 contacts the Hoogsteen face of the 1542C:1550G (m.3212C:3230G) base pair. MRPL32/bL32 contacts the ribose of 1542C (m.3212C) and establishes additional contacts to MRPL3/uL3m. MRPL17/uL17 also establishes contacts to the RNA backbone at position 1544C (m.3214C). Protein MRPL39/mL39 establishes contacts to all three mito-ribosomal proteins that contact H100, namely MRPL3/uL3m, MRPL17/uL17, and MRPL32/bL32, and to the mito-ribosomal tunnel protein MRPL22/ul22, further away. Mutations in MRPL3/uL3m have been identified in association with cardiomyopathy, neonatal lactic acidosis, sensorineural hearing loss, cirrhosis, and interstitial nephritis (Bursle et al., 2017, Galmiche et al., 2011. In addition, proper maturation of MRPL32/bL32 has been shown to be crucial for mitochondrial translation in yeast and in mammals (Nolden et al., 2005). Interactions are well supported by density. All this evidence indicates an important structural role for H100 as a hub for the proper folding of the mito-ribosomal LSU. While the 1543A>G (m.3213A>G) variant would replace an A:U Watson-Crick base pair with a disfavored G•U wobble at the beginning of a helical segment (Ananth et al., 2013). Hence, it is possible that the substitution could slightly alter the structure of the region, thus affecting the assembly of the subunit. In this light, the potential pathogenicity of the 1543A>G (m.3213A>G) variant cannot be ruled out.

Unclear variants -220C>U (m.867C>U):
The 220C>U (m.867C>U) variant was found in a patient with sensorineural hearing loss (Guaran et al., 2013). In the secondary structure of 12S mt-rRNA, position 220C (m.867G) lies in the single-stranded stretch connecting helices h4 and h18. The O2 of 220C (m.867G) has been modeled sharing a hydrogen bond with the amino group of 223C (m.870C), also within the same rRNA stretch (not shown). However, the quality of the density in this region cannot be used to confirm the accuracy of this interaction. This region is recognized by the protein MRPS18B/bS18b. No conclusions can be drawn from the data.

-380A>G (m.1027A>G):
The 380A>G (m.1027A>G) variant was identified by Lu et al. (Lu et al., 2010). Position 380A (m.1027A) is located in the GNRA tetraloop capping h23a. A bifurcated sheared base pair between 377G (m.1024G) and 380A (m.1027A) closes the GNRA tetraloop (Correll Carl C. et al., 1998). While the structure of this base pair should not be disrupted by the A>G substitution, the C2 of 380A (m.1027A) lies only 3.7 Å away from the RNA backbone at position 406A (m.1053A), suggesting that the presence of a bulky 2-amino group, as a result of the A>G base change, could be disruptive. Since the GNRA tetraloop docks into the minor groove of h20 via a conserved double A-minor interaction mediated by positions 378-9A (m.1025-6A) (Noller., 2005), a small disruption of the local structure could have ampler effects.

Supplementary Figures
All sites of variation discussed in the main text are shown here in the context of the secondary structure of 12S mt-rRNA, as well as in the higher-order structure context of the mitoribosome. All Suppl. Figures were created with Chimera X (Pettersen et al., 2021). Most of the structural data was obtained with the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB Protein Data Bank ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022. All other structures are mentioned in the legends. At least one panel is used to show the agreement between the structural model and the experimental electron density. Contour levels were adjusted to display the shape of RNA residues. In cases in which the chosen contour level did not cover certain features of interest in the model, a second panel at a lower contour level might be included in the Suppl. Figure. Supplementary Figure S1 molecules are shown as red spheres. D-E. Chimera X-rendered electron density (black mesh) at a contour level of 0.032 (D) and 0.022 (E) (Pettersen et al., 2021). MRPS12/uS12m is shown in light blue in E. The molecular model and electron density map from the 2.2-Å cryo-EM human mito ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-E.

Supplementary Figure S2. Positions 139G (m.786G) in the human mito-ribosome. A.
Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair, thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map. B. Annotated view of the region containing the 139G>A (m.786G>A) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. The position of K43 of MRPS12/uS12m is indicated in B and D. Hydrogen bonds are indicated by blue, broken lines. MRPS12/uS12m is shown as a grey surface. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.042 (C) and 0.022 (D) (Pettersen et al., 2021). MRPS12/uS12m is shown in light blue in D. The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S3
Supplementary Figure S3. Positions 178U (m.825U) and 180A (m.827A) in the human mito-ribosome. A. Localization of the variantcontaining region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-" canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 178U>A (m.825U>A) and 180A>G (m.827A>G) variants (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. MRPS12/uS12m is shown as a grey surface. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.032 (C) and 0.022 (D), the latter used to demonstrate the existence of clear electron density around MRPS12/uS12m residues (Pettersen et al., 2021). MRPS12/uS12m is shown in light blue in C and D. The positions of R55 and S86 of MRPS12/uS12m are indicated in C. E. Position of EFG1 (magenta surface), relative to 12S rRNA. Distances are denoted with black, broken lines. The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D. The EFG1 structure was obtained from RCSB: 6VMI (Koripella et al., 2020) and superimposed onto the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 with the ChimeraX Matchmaker utility, using MRPS12 as the reference chain (Pettersen et al., 2021). Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; " •", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 295A>G (m.942A>G), 400A>G (m.1047A>G), and 401C>U (m. 1048C>U) variants (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled and color coded to their molecular model. The position of NAD (heteroatom coloring) is indicated. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. MRPS12/uS12m is shown as a grey surface. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.042 (C) and 0.022 (D), the latter used to demonstrate the existence of clear electron density around the water molecules mentioned in the text (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S5
Supplementary Figure S5. Position 304G (m.951G) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 304G>A (m.951G>A) variant (yellow and labeled in bold, black font). Other rRNA residues are labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. MRPS12/uS12m is shown as a grey surface. C. Chimera Xrendered electron density (black mesh) at contour level of 0.044 (Pettersen et al., 2021). The positions of N34 of MRPS12/uS12m and N44 of MRPS17/uS17 are indicated. The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B and C.

Supplementary Figure S6
Supplementary Figure S6. Positions 459C (m.1106C) and 460U (m.1107U) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions (central domain pseudoknot). B. Annotated view of the region containing the 459C>U (m.1106C>U) and 460U>C (m.1107U>C) variants (yellow and labeled in bold, black font). Other rRNA residues are labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled and color coded to their molecular model. Distances are denoted with black, broken lines. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.052 (C) and 0.022 (D) (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 76A>C (m.723A>C) and 145C>U (m.792C>U) variants (yellow and labeled in bold, black font). Other rRNA residues are labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.03 (C) and 0.02 (D), the latter used to better resolve the electron density around MRPS25/mS25 (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S8
Supplementary Figure S8. Positions 95U (m.742U) and 98A (m.745A) in the human mito-ribosome. A. Localization of the variantcontaining region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 95U>C (m.742U>C) and 98A>G (m.745A>G) variants (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.03 (C) and 0.02 (D), the latter panel focused on the interaction between h7 and MRPS16/bS16 (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S9
Supplementary Figure S9. Position 209A (m.856A) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 209A>G (m.856A>G) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.031 (C) and 0.021 (D) (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D. EFG1 is shown as a magenta surface. The EFG1 structure was obtained from RCSB: 6VMI (Koripella et al., 2020) and superimposed onto the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 with the ChimeraX Matchmaker utility, using MRPS12 as the reference chain (Pettersen et al., 2021).  (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S11
Supplementary Figure S11. Position 878C (m.1525C) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 878C>G (m.1525C>G) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.020 (C) and 0.012 (D), the latter used to demonstrate the existence of clear electron density around the variant position (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021) were used to create panels B-D. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 0.025 (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S13
Supplementary Figure S13. Position 596U (m.1243U) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 596U>C (m.1243U>C) variant (yellow and labeled in bold, black font). Other rRNA residues are labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. The position of R36 of MRPS14/uS14m is indicated. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 5.88 (Pettersen et al., 2021). The molecular model and electron density map from the 2.59-Å cryo-EM human mito-ribosomal structure (RCSB ID: 6ZM6) (Itoh et al., 2021) were used to create panels B-D.

Supplementary Figure S14
Supplementary Figure S14. Position 663C (m.1310C) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 663C>U (m.1310C>U) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 0.032 (C) and 0.022 (D) (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S15
Supplementary Figure S15. Position 735A (m.1382A) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 735A>C (m.1382A>C) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled and color coded to their molecular model. The position of R166 of MRPS29/mS29 is indicated. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 0.03 (C) and 0.018 (D), the latter used to demonstrate the existence of clear electron density around protein MRPS7/uS7m (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.

Supplementary Figure S16
Supplementary Figure S16. Position 796U (m.1443U) in the human mito-ribosome. A. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; "•", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 796U>C (m.1443U>C) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. The position of R102 of MRPS14/uS14m is indicated. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 0.0375 (Pettersen et al., 2021). D. Chimera X-rendered electron density (black mesh) at contour level of 5.88 (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B and C. The molecular model and electron density map from the 2.59-Å cryo-EM human mito-ribosomal structure (RCSB ID: 6ZM6) (Itoh et al., 2021) were used to create panel D. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; " •", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 791G>A (m.1438G>A), 805U>C (m.1452U>C), and 806A>G (m.1453A>G) variants (yellow and labeled in bold, black font). Other rRNA residues are labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C. Chimera X-rendered electron density (black mesh) at contour level of 0.032 (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D. Localization of the variant-containing region in the secondary structure map of 12S mt-rRNA. Sites of variation are labeled in magenta. Additional sites are labeled in black. Helix numbers are shown in light blue. Symbols: "-", canonical base pair; " •", wobble base pair; "•", non-canonical base pair; thick, black line, physical connection and continuity between adjacent bases that are drawn distantly in the secondary-structure map; red squares connected by red, thick lines, tertiary interactions. B. Annotated view of the region containing the 469A>G (m.1116A>G), 471A>G (m.1118A>G), and 472U>C (m.1119U>C) variant (yellow and labeled in bold, black font). Other rRNA residues labeled in regular font. 12S mt-rRNA is shown in grey with helix numbers indicated. Other components of the mito-ribosome are labeled, and color coded to their molecular model. The positions of R100 of MRPS2/uS2m, R297 of MRPS5/uS5m, and R50 of MRPS23/uS23 are indicated. Hydrogen bonds are indicated by blue, broken lines. Distances are denoted with white, broken lines. Water molecules are shown as red spheres. C and D. Chimera X-rendered electron density (black mesh) at contour level of 0.035 (C) and 0.025 (D), the latter used to demonstrate the existence of clear electron density around position 469A (m.1116A) (Pettersen et al., 2021). The molecular model and electron density map from the 2.2-Å cryo-EM human mito-ribosomal structure (RCSB ID: 8ANY) (Itoh et al., 2021, Itoh et al., 2022 were used to create panels B-D.