Naked mole-rat has increased translational fidelitycompared with the mouse, as well as a unique 28Sribosomal RNA cleavageJorge Azpuruaa,1, Zhonghe Kea,1, Iris X. Chena, Quanwei Zhangb, Dmitri N. Ermolenkoc, Zhengdong D. Zhangb,Vera Gorbunovaa,2, and Andrei Seluanova,2

aDepartment of Biology, University of Rochester, Rochester, NY 14627; bDepartment of Genetics, Albert Einstein College of Medicine, New York, NY 10461;and cDepartment of Biochemistry and Biophysics, University of Rochester, Rochester, NY 14642

Edited* by Eviatar Nevo, Institute of Evolution, Haifa, Israel, and approved September 3, 2013 (received for review July 18, 2013)

The naked mole-rat (Heterocephalus glaber) is a subterranean euso-cial rodent with a markedly long lifespan and resistance to tumor-igenesis. Multiple data implicate modulation of protein translationin longevity. Here we report that 28S ribosomal RNA (rRNA) of thenaked mole-rat is processed into two smaller fragments of un-equal size. The two breakpoints are located in the 28S rRNA di-vergent region 6 and excise a fragment of 263 nt. The excisedfragment is unique to the naked mole-rat rRNA and does not showhomology to other genomic regions. Because this hidden breaksite could alter ribosome structure, we investigated whether trans-lation rate and amino acid incorporation fidelity were altered. Wereport that naked mole-rat fibroblasts have significantly increasedtranslational fidelity despite having comparable translation rateswith mouse fibroblasts. Although we cannot directly test whetherthe unique 28S rRNA structure contributes to the increased fidelityof translation, we speculate that it may change the folding ordynamics of the large ribosomal subunit, altering the rate of GTPhydrolysis and/or interaction of the large subunit with tRNA dur-ing accommodation, thus affecting the fidelity of protein synthe-sis. In summary, our results show that naked mole-rat cellsproduce fewer aberrant proteins, supporting the hypothesis thatthe more stable proteome of the naked mole-rat contributes toits longevity.

aging | NMR

Naked mole-rats (NMRs; Heterocephalus glaber) are extremelongevity outliers compared with other rodents of their size.Although only slightly larger than a laboratory mouse, the NMRlives an order of magnitude longer, with a maximum recordedlifespan of more than 30 y for both reproductive and non-reproductive castes (1). It was also noted that NMRs aremarkedly resistant to the increased frailty that accompanies ag-ing in most metazoans. Furthermore, despite observations ofmany aged individuals, no neoplasia has been reported in theNMR (2, 3).NMR cells have been experimentally shown to be much more

resistant to oncogenic transformation than mouse cells (4, 5).The mechanism of tumor resistance is mediated by hypersensi-tivity of NMR cells to contact inhibition triggered by hyaluronanof extremely high molecular mass (5–7). Although these studiesexplain why the animals are tumor-resistant, they do not explaintheir overall longevity and their ability to maintain somatic in-tegrity until extreme age. Previous studies have indicated that theNMR is able to tolerate high intracellular levels of oxidizedproteins and oxidized lipids (8–10). Furthermore, the NMR isnotably able to maintain proteomic integrity (as evidenced bylower protein unfolding after treatment with urea) until very latein its lifespan without an age-dependent increase in proteinubiquitination, such as observed in short-lived mice (11).Multiple signaling pathways implicated in aging and cancer

converge on factors that modify the rate of translation initiationand elongation (12–15). One of the main functions of the mTOR

pathway is the promotion of protein translation in response tonutrients. Modulating the mTOR pathway genetically or phar-macologically, using the inhibitor rapamycin, has been shown toextend lifespan in yeast, Caenorhabditis elegans, Drosophila, andmice (14). The exact mechanism by which modulating proteintranslation via inhibition of mTOR extends lifespan is unclear.Two major explanations that were proposed are global reductionin mRNA translation, leading to better maintenance of proteinhomeostasis, or differential translation of specific mRNAs ben-eficial for longevity and stress resistance (reviewed in ref. 13).Proper protein folding and stability are heavily implicated in

aging. Overexpression of heat shock transcription factors andchaperones that aid protein folding can extend lifespan in Dro-sophila melanogaster (16) and C. elegans (17, 18). In bacteria,aging occurs by asymmetrical segregation of protein aggregates(19), and a similar mechanism has been described in yeast (20).The formation of aggregates as a result of mistranslated poly-peptides has been implicated in several aging-related diseasessuch as amyotrophic lateral sclerosis, Huntington’s disease, andAlzheimer’s disease (21). A major factor determining proteomequality is translational fidelity of the ribosome. Severe disruptionsof translational fidelity have been shown to cause accumulationof aggregates and pathological neurodegeneration (22).The connection between translation and aging led us to ex-

amine NMR ribosomes for longevity-promoting phenotypes suchas altered translation rate and translational fidelity. We describea unique ribosomal DNA insertion, which is processed afterrRNA transcription and breaks the NMR 28S rRNA into twodistinct fragments. We assayed ribosomal translation rate (com-bined initiation and elongation) by measuring bulk incorporation

Significance

Molecular mechanisms responsible for differences in longevitybetween animal species are largely unknown. Here we showthat the longest-lived rodent, the naked mole-rat, has moreaccurate protein translation than the mouse. Furthermore, weshow that the naked mole-rat has a unique fragmented ribo-somal RNA structure. Such cleaved ribosomal RNA has beenreported for only one other species of mammal. This articlesuggests the importance of protein translation in aging andprovides insight into the mechanisms of longevity.

Author contributions: J.A., Z.K., Z.D.Z., V.G., and A.S. designed research; J.A., Z.K., I.X.C.,Q.Z., and Z.D.Z. performed research; J.A., Z.K., Q.Z., D.N.E., Z.D.Z., V.G., and A.S. analyzeddata; and J.A., Z.K., Z.D.Z., V.G., and A.S. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1J.A. and Z.K. contributed equally to this work.2To whom correspondence may be addressed. E-mail: [email protected] [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1313473110/-/DCSupplemental.

17350–17355 | PNAS | October 22, 2013 | vol. 110 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1313473110

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of [35S]-cystein/methionine into nascent polypeptide chains. Wefound that NMR and mouse skin fibroblasts have roughly equalrates of translation. By using firefly luciferase reporters witha variety of mutations, including amino acid substitutions or stopcodons that required misincorporation or frameshift to occur toreactivate the luciferase, we found that primary NMR fibroblastshave a fourfold lower rate of amino acid misincorporation thanprimary mouse fibroblasts. These results suggest that NMR cellsproduce fewer aberrant proteins and support the hypothesis thatthe NMR’s stable proteome is a contributor to its longevity.

ResultsNMR 28S rRNA Has an Atypical Pattern. During routine extraction ofRNA, we noticed that the 28S ribosomal RNA of the NMR wasprocessed into two fragments of unequal size. NMR RNAextracts from several tissues (liver, lung, brain, spleen, kidney,and testis) exhibited an unusual rRNA band pattern, whereasmouse RNA extracts did not (Fig. 1A). We observed this patternafter using two different methods of RNA extraction in extractsprepared from tissues and cultured cell lines (Fig. S1A). We alsoextracted RNA from other nonmodel rodents (Fig. S1B) andfound the break to be unique for the NMR.To confirm that the break was not a result of RNase

contamination, we combined NMR cells with mouse cells andsimultaneously extracted RNA after homogenizing them. Whenindependently extracted, the fibroblast RNA showed three bandsand two bands for NMR and mouse, respectively. In the mixedsample, all bands could be seen, indicating that no nucleasecontamination was cleaving the mouse rRNA (Fig. 1B). ThisNMR 28S rDNA was undergoing specific processing in vivo.

Mapping of the 28S rRNA Cleavage Sites. To determine the site andsequence of the NMR 28S rRNA breakage point, we firstdesigned primers to conserved 28S rDNA sequence from themouse genome. We estimated the location of the breakpointregion by noting that the sizes of the two fragments (2.5 and

3 kb) were only slightly different, meaning the break would bewithin ∼1 kb of the center. This allowed us to amplify a central1-kb fragment of genomic NMR 28S rDNA. This fragmentincluded 28S divergent region 6 (D6) that exhibited a 118-ntunique insertion compared with the mouse sequence flankedby regions of weak homology with the mouse D6 (Fig. 2).Using the 1-kb sequence and a publically available NMRcoding DNA sequence, we designed primers to perform 5′RACE and 3′ RACE. After aligning with the NMR 28S rDNAsequence, we identified the two cleavage sites and found thata 263-nt fragment flanked by 5-nt direct repeats 5′-CGGAC-3′was cleaved out in the 28S rRNA, leaving behind the fragmented28S rRNA. The 263-nt fragment included the 118-nt insertionthat was not homologous to the mouse sequence (Fig. 2). Theribosomal DNA loci are repeated thousands of times, and fromour RNA extractions, most of the products seemed to be cleaved.We used a bioinformatics approach to test whether the identifiedcut sites were conserved among all of the 28S rDNA loci in theNMR. From whole-genome shotgun reads from two differentNMR genome sequences [Broad Institute (http://www.ncbi.nlm.nih.gov/nuccore/AHKG00000000) and Kim et al. (23)], the cutsites were found to be conserved among all of the rDNA loci (Fig.S2 and Table S1), which confirmed that the majority of 28SrRNAs are cleaved in the NMR.

NMR Translation Rate Does Not Differ from the Mouse. We hy-pothesized that the fragmented 28S rRNA might cause alternateassembly or structure of the NMR ribosome, with detectableconsequences for protein synthesis rate or fidelity. To investigatewhether the NMR ribosome had a different translation rate than

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Fig. 2. Structure of the NMR 28S rRNA and localization of break sites. (A)Overall structures of the 28S rRNA in mouse, NMR, and Ctenomys. Themouse 28S rRNA has no cleavage sites. The D6 region of the NMR 28S rRNAcontains a unique 118-nt insertion (shaded box). The two cleavage sites thenexcise a 263-nt fragment. Ctenomys is the only other known mammal withfragmented rRNA. The D6 region of the Ctenomys 28S rRNA contains aunique 106-bp insertion (shaded box) with the cleavage site located withinthis unique sequence. Thick black lines represent the 28S rRNA, the whitebox represents the D6 region, the arrows denote the cleavage sites, and thesize of the D6 region and the distance between the two cleavage sites areindicated. (B) The alignment of the NMR and mouse 28S rDNA sequences.The box highlights the D6 region, the arrows indicate the cleavage sites, andhyphens indicate the gap.

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the mouse ribosome, we implemented an assay based on mea-suring [35S]-cystein/methionine incorporation into nascent pro-tein. As the physiological temperature of the NMR is 32 °C,rather than 37 °C, and NMR fibroblasts are usually cultured at32 °C, we tested the translation rate at both temperatures forboth mouse and NMR cells. Mouse and NMR fibroblasts weregiven a pulse of [35S]-cystein/methionine for 30 min, cultured at32 °C and 37 °C, total cell proteins were extracted, and theamount of radioactive isotope incorporation was measured bya scintillation counter.The translation rate was found to be altered twofold by shifting

the temperature by 5° in both species (Student t test, NMR, P =0.02; mouse, P = 0.0027), but no significant differences were seenbetween the NMR and mouse cells when they were temperature-matched (Fig. 3A). To confirm that the protein quantification wasaccurate and that mouse and NMR fibroblasts have similaramounts of ribosomes, we performed Western blots on ribosomalprotein S6 (RPS6). No large differences were seen in the overallamount of ribosomal protein or loading controls from mouse andNMR fibroblasts (Fig. 3B). Taken together, these results show thatthe NMR translation rate is equivalent to that of the mouse whenthey are temperature-matched, and that the cells express similarlevels of a ribosomal protein.

NMR Has Higher Translational Fidelity than the Mouse. We next ex-amined whether altered ribosomal structure might affect theoverall translation fidelity of proteins. To assay mouse and NMRcells for translational fidelity, we constructed a set of firefly lu-ciferase mutant reporters. In these reporters, a key amino acid inthe luciferase protein (lysine 529) was mutated to completelydestroy luciferase activity (24), and only if the wrong amino acidwere incorporated into the nascent polypeptide would enzymaticactivity be restored. The reporters included all triplet positions,

premature stop (third codon mutation at the beginning of theprotein), and both positive and negative frameshifts by nucleo-tide insertion and deletion, respectively.The luciferase assay indicated that NMR cells had higher

protein translation fidelity than mouse cells. This was particularlynoticeable when mouse and NMR cells were grown at 32 °C, asthe NMR cells had ∼10-fold lower misincorporation frequencyfor the lysine triplet mutants, and these differences were highlystatistically significant for all reporters (Student t test, P < 0.005)(Fig. 4A). At 37 °C, NMR cells had approximately a fourfoldlower misincorporation; this was also statistically significant forall reporters (Student t test, P < 0.05) (Fig. 4B).Notably, the NMR cells were able to maintain a high trans-

lational fidelity even when forced to grow more quickly. We in-tegrated simian virus 40 (SV40) Large T-antigen (LargeT) andhuman Ras Glycine 12 to Valine (H-RasV12) mutant proteininto wild-type NMR fibroblasts to induce more rapid growth viaconstant Ras signaling and abrogation of the p53/retinoblastomaprotein pRB tumor suppressor pathways to prevent oncogene-induced senescence. The fidelity assays were then repeated onthese cells grown at 32 °C and 37 °C. The translation error ratewas not significantly different from in the primary NMRfibroblasts at either temperature (Fig. 4 A and B), indicating thateven when NMR cells proliferate at a similar rate as mouse cells,they still maintained higher translational fidelity. This resultsuggests the NMR ribosomes are intrinsically more accurate thanthe mouse ribosomes.

DiscussionOur study shows an unexpected processing of NMR 28S rRNAinto two molecules of 2.5 and 3 kb, corresponding to 3′ and 5′segments of the rRNA gene. The break occurs within the D6region and results from two cuts that excise a 263-nt fragmentcontaining a unique 118-nt sequence. This 118-nt sequence bearsno significant homology to other known sequences. The cleavageoccurs in all NMR organs that we analyzed, as well as in theprimary fibroblast cultures.The excised fragment is flanked by 5-nt direct repeats, which

supports that the insert originated from a transposable element.The sequences distal to the cut sites share weak homology withthe mouse D6, suggesting that the insert could have also beenintroduced by homologous recombination. We do not know themechanism responsible for the cleavage of the NMR 28S rRNA.The cuts could be generated by a ribozyme activity residingwithin the D6 sequence itself, as was reported for R2 trans-posons residing within the rDNA locus (25). We tested thispossibility by in vitro transcribing 28S rRNA; however, the invitro transcribed 28S rRNA molecules remained uncleaved. Fi-nally, the cleavage may be mediated by a ribonuclease that rec-ognizes the D6 region.Fragmented rRNA has been found in Salmonella (26), pro-

tozoa (27, 28), a worm (29), and several arthropods (ref. 30 andreferences therein). However, in vertebrates there is only oneother example of a fragmented rRNA, found in rodents of thegenus Ctenomys, also known as tuco-tuco (31). In Ctenomys, 28SrRNA is cleaved within the D6 region, but the cleavage occurs withonly a single cut site, and sequences surrounding the break share nohomology with the NMR sequence (Fig. 2A). Interestingly, Cten-omys is a South American rodent that is phylogenetically distantfrom the NMR but shares similar ecology. Ctenomys is a sub-terranean rodent, with some species of the genus leading a sociallifestyle. The maximum lifespan in captivity is not determined forCtenomys, but in the wild, Ctenomys is short-lived, with an averagelifespan of 3 y (32).We found that NMR cells had equivalent translation rates

(adjusted for physiological temperature). However, by construct-ing a set of unique luciferase reporters, we observed that NMRcells have substantially higher translation fidelity than mouse cells,

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Fig. 3. NMR and mouse have similar translation rates. (A) [35S]-cystein/methionine incorporation assay performed on growing fibroblasts showingNMR translation occurs at roughly equal speed to that of mouse fibroblasts.The assays were performed at 32 °C and 37 °C for both mouse and NMR cells.NMR SF are primary NMR skin fibroblast and MSF are primary mouse skinfibroblast. (B) Western blot against RPS6, a protein component of the ma-ture ribosome (Upper) and β-actin as loading control (Lower).

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even when transformed with oncogenes to increase their cell cyclespeed. NMR cells also displayed higher fidelity of translation forall errors we tested, including misincorporation, stop codon skip-ping, and frameshifts. A potential caveat of our translation fidelityassay is that active firefly luciferase could be generated not only byincorrect translation but also by incorrect transcription or randommutations of the plasmid DNA during cell growth. This seemsunlikely given that if replication or transcription produced activeluciferase, we would not see the expected nucleotide position biasthat the assay clearly shows (with the third position being the leastmistranslated). Furthermore, we would not expect to see sucha large bias in favor of insertions over deletions. The mis-incorporation rate we detect in our assay is also between 1:1,000and 1:10,000, which is consistent with reports in the literature (24).Thus, we conclude that most of the luminescence from our assaycomes from mistranslation, rather than from mistakes at thenucleotide level.Pérez et al. reported that the NMR proteome has higher levels

of cysteine than the mouse proteome and that the proteins areresistant to unfolding stressors such as 1 M urea. These resultsare consistent with high levels of translation fidelity (11).According to our results, the mouse proteome would have two-to 10-fold higher levels of proteins with misincorporated aminoacids relative to the NMR. These misincorporated amino acidscould increase the number of proteins that are unstable underchallenging conditions, such as urea treatment. Pérez et al. alsoreport that the NMR does not show an age-dependent increasein protein ubiquitination. This is consistent with the high fidelityof protein translation in the NMR. More recently, it has beenreported that NMR proteins maintain a higher level of activityafter carbonylation and oxidation than the equivalent mouseproteins (33). Again, it is possible that fewer amino acid mis-incorporations allow proteins to tolerate more stochastic impropermodifications and oxidative damage, and thus retain tertiary struc-ture and activity.Given the remarkable longevity of the NMR, it is possible that

increased translational fidelity plays a role in its longevity. The“error catastrophe theory of aging” first proposed by Orgel in1963 (34) and explored in Escherichia coli by Edelmann andGallant in 1977 posits that early defects in translational fidelitylead to a vicious cycle of progressively more severe defects inprotein homeostasis (35). Differences in translational fidelitymay play an important role in determining lifespan. Many age-related disorders are caused by protein aggregation (21), andproblems with translational fidelity can lead to the accumulationof misfolded protein and the formation of inclusion bodies (22).The proteome is maintained by various mechanisms, all of whichshow an age-dependent decline (36). During heat stress, chaper-ones are up-regulated, but this mechanism fails with age (37, 38).Proteasome activity also declines with age (39). Aging also leads toa decline in macroautophagy, which eliminates misfolded or in-correctly glycosylated proteins from the cell (40). It is possible thatwhen proteome maintenance mechanisms fail, it allows the aberrantmistranslated proteins to accumulate. Species, such as the NMR,with a lower overall level of mistranslated proteins would thus bebuffered against some of the decline of old age, given that they havefewer misfolded proteins to begin with.The main source of mistranslated proteins in E. coli is discrimi-

nation of noncognate or near-cognate tRNAs during translation(24). The ribosome structure itself confers at least some of thefidelity of the process, as shown by hyperaccurate ribosomes (41),and its sequence and structure (42) may be altered by natural se-lection in species that evolve long lifespans. Indeed, in E. coli, thereis an observed evolutionary tradeoff between ribosomal speed andaccuracy (41). Because the NMR in vitro translation system cur-rently cannot be reconstituted from purified components, we areunable to directly test whether 28S cleavage is responsible for im-proved translation fidelity in the NMR. The cleavage leads to

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Fig. 4. NMR has higher fidelity of translation than the mouse. Translationfidelity of NMR and mouse skin fibroblasts was quantified as a frequency ofamino acid misincorporation into a set of firefly luciferase reporters witha mutated critical lysine 529 (Fig. S3). Misincorporation events can rescueluciferase activity. Luminescence of mutated firefly luciferase was measuredand normalized to the luminescence of Renilla luciferase, both under controlof the CMV promoter. The positive control (wild-type luciferase) ratio wasseveral orders of magnitude higher and is cropped for visibility. The mutantreporters E – K529E; I – K529I; N – K529N measured misincorporation into thefirst, second, and third codon positions, respectively; the STOP reportercontained a premature stop codon mutation at amino acid 81; FS+ con-tained a positive frameshift mutation by nucleotide insertion at position 81;and FS contained a negative frameshift mutation by nucleotide deletion atposition 81. Results for the assay are shown for NMR and mouse cells grownat 32 °C (A) and 37 °C (B). Error bars show standard deviation. Asterisks in-dicate values significantly different between NMR and mouse (P < 0.005)Student t test.

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deletion of the major part of the D6 region corresponding toa helix 45 of 23S rRNA in E. coli and leaves two fragments of28S rRNA disconnected. This may change the folding or dy-namics of the large ribosomal subunit, altering the rate of GTPhydrolysis by elongation factor 1A and/or interaction of thelarge subunit with tRNA during accommodation, thus affect-ing the fidelity of protein synthesis. In particular, in bacterialribosome, helix 45 of 23S rRNA is connected to the L11-stalk(L12 stalk in mammals), which is formed by helices 42, 43, and44 through coaxial stacking with helices 40 and 41. Thus, thedeletion of helix 45 may affect dynamics of the L11 stalk,which directly interacts with the elbow of the aminoacyl-tRNAin the intermediate (A/T) state of tRNA accommodation (43).In summary, we have shown that NMR has a unique form of 28S

rRNA that is processed in two fragments. The processing occurs bytwo cuts within the D6 region. Furthermore, we demonstrate thatNMR has higher fidelity of translation than the mouse. We hy-pothesize that the unique structure of the NMR 28S rRNA maycontribute to the higher fidelity of translation, which in turn maycontribute to exceptional longevity of the NMR.

Materials and MethodsRNA Extraction and Denaturing Gel Electrophoresis. RNA from rodent tissuesand cells was extracted either by RNEasy Kit (Qiagen), according to themanufacturer’s instructions, or by TriReagent extraction. TriReagent ex-traction was performed by mixing cells or tissues with TriReagent, rupturingin a glass homogenizer, and centrifuging at high speed (>10,000 × g) at 4 °Con a tabletop centrifuge. The aqueous phase was then washed with iso-propanol to precipitate RNA and then washed with ethanol, dried, andresuspended in RNase-free water.

Denaturing agarose gels were prepared in double-distilled H2O and20× Mops buffer diluted to 1× concentration. Formaldehyde was addedto a final concentration of 2% (vol/vol). RNA samples were denatured informaldehyde/Mops buffer at 80 °C for 5 min and then immediatelyplaced on ice for at least 2 min. Samples were loaded and run at ∼100 Vfor 2–3 h.

rRNA Break Mapping. The unbroken 28S rRNA was estimated to be ∼5 kbp,and because the two fragments were close in size, we estimated that thebreak probably happened within 1,000 bp of the middle. 5′ RACE primersspecific to highly conserved regions downstream of the putative 28S mid-point were designed. A 5′/3′ second-generation RACE kit (Roche) was usedto obtain a fragment that mapped from the conserved region to the 5′break point. Primers were designed that mapped upstream of the putativebreakpoint, and 3′ RACE was performed by ligating an adenylated linker tothe 3′ end of the ribosomal RNA. PCR was then used to extend from theupstream conserved site to the start point of the break. Primer sequencesare shown in Table S2.

Tissue Culture. NMR fibroblasts were grown at 32 °C (in vivo body temperature,unless otherwise indicated) 5% CO2, 3% O2 on treated polystyrene culturedishes (Corning) in eagle’s minimum essential medium (EMEM) media (ATCC)supplemented with 15% FBS (Gibco), nonessential amino acids, sodium pyru-vate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Mouse cellswere grown in identical conditions except at 37 °C, unless otherwise indicated.

Translation Rate Assay. Cells were grown to ∼75% confluence, and media wasremoved and incubated for 15 min with cysteine/methionine-free media todeplete intracellular pools. The media was then removed, and cells were in-cubated for 30 min in media containing [35S]-methionine/cystein. Radioactivemedia was then removed, and cells were incubated with 1.5 mL ice-cold lysisbuffer and scraped from the plate, using a disposable plastic scraper. The cellswere then stored at −80 °C overnight. Trichloroacetic acid (TCA) precipitationwas used the following day to pellet proteins, and the pellet was washed with

acetone. Lowry assay was performed to determine protein concentration.Radioactivity was evaluated by scintillation counter (Beckman).

Western Blot. Proteins samples were run through 15% SDS/PAGE and transferredto nitrocellulose via the TransBlot Turbo system (BioRad). The membrane was cutand blotted separately for ribosomal protein RPS6 and beta-actin (loadingcontrol). The membrane was incubated with primary antibody overnight at 4 °Cand with secondary antibody for 1 h at room temperature. Antibodies usedwere Santa Cruz biotechnology SC-47778 (beta-actin) and Cell Signaling2317 (RPS6).

Plasmid Construction and Transfection. pGL3 wild-type luciferase plasmid(Promega) was mutated as indicated by site-directed mutagenesis, usinga QuikChange kit (Agilent). Several clones were picked, miniprepped, and sent forsequencing to confirm proper mutagenesis. Correct plasmids were prepped usingEndoFree Maxi prep kit (Qiagen). Cells were thawed and grown for two passagesbefore being harvested. Cells were counted, and 1 × 106 cells were transfectedusing Nuclefector transfection (Lonza) with program U-020 in human dermalfibroblast (NHDF) solution with 5 μg reporter plasmid [mutant, wild-type-positivecontrol, or hypoxanthine-guanine phosphoribosyltransferase (HPRT) negativecontrol] and 0.1 μg Renilla luciferase plasmid (transfection/promoter control).Cells were then pipetted onto 10 cm Corning treated plates and allowed to growfor 24 h at the indicated temperature. Mutation sites within Luciferase gene areshown in Fig. S3.

Luciferase Assay. A Promega Dual-Luciferase assay kit was used to performanalysis on transfected cells. Cells were harvested, counted, and lysed, usingthe provided buffer. Extracts were thoroughly mixed with LAR-II reagent, andluminescence was measured. The sample was then mixed with STOP&GLOsolution (halting firefly luciferase and starting Renilla luciferase), and lumi-nescence was measured. Ratio of firefly to Renilla was used as an indicatorof translational fidelity.

Assembly of 28S rRNA Sequence from Illumina Raw Reads. First, we mapped thepaired-end reads to the mouse 28S DNA sequence, using Bowtie2 (44), whichallows mismatches and small indels/gaps. Then Velvet (45) was used to as-semble the reads that are mapped to the mouse 28S DNA sequence. The set ofcontigs with the largest N50 were selected. However, it is hard to obtain thereads from divergent regions by this method. To find scaffolds that the coverNMR 28S gene, we mapped our contigs to all of the scaffolds obtained bygenome-wide assembly. Finally, we find one scaffold that matched our contigsthe most.

Divergence Analysis of the Cleavage Sites in the D6 Region. To assess the di-vergence/conservation at the two cutting sites in D6 among all of the copies of 28Sin NMR, we used the following strategy: Given a single sequenced copy of NMRD6 and its flanking regions, the conserved 50-bp sequences immediately upstreamto the start of D6 and downstream to the end of D6 were used as the target forthe 5′ and 3′ end cutting sites, separately. We mapped the target to the readswith the maximum 3 mismatch (i.e., we take the sequencing short reads as thereferences and do alignment for the target). After we get the matched reads, ifthe read covers the cutting site, we save the subsequence around the cuttingsite. We kept ±15 bp from the 5′ end cutting site and ±18 bp from the 3′ endcutting site, by which we centered the cutting site in the subsequencesaround the cutting sites. Finally, the multiple sequence alignment programClustal Omega (46) was used to align the subsequences and the nucleotidefrequency was counted based on the multiple sequence alignment result. Fig.S2 shows the divergence analysis strategy. Table S1 shows that nucleotidefrequency at the two cutting sites. The cutting sites were highly conservedamong the 28S rRNA copies.

ACKNOWLEDGMENTS. We thank members of the T. Eickbush and G. Culverlaboratories at the University of Rochester for useful discussion and sugges-tions. This work was supported by the National Institutes of Health andEllison Medical Foundation grants (to V.G.) and by Life Extension Foundationgrants (to V.G. and A.S).

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