METTL1/WDR4-mediated m7G tRNA modifications and m7G codon usage promote mRNA translation and lung cancer progression

Mis-regulated epigenetic modifications in RNAs are associated with human cancers. The transfer RNAs (tRNAs) are the most heavily modified RNA species in cells; however, little is known about the functions of tRNA modifications in cancers. In this study, we uncovered that the expression levels of tRNA N7-methylguanosine (m7G) methyltransferase complex components methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) are significantly elevated in human lung cancer samples and negatively associated with patient prognosis. Impaired m7G tRNA modification upon METTL1/WDR4 depletion resulted in decreased cell proliferation, colony formation, cell invasion, and impaired tumorigenic capacities of lung cancer cells in vitro and in vivo. Moreover, gain-of-function and mutagenesis experiments revealed that METTL1 promoted lung cancer growth and invasion through regulation of m7G tRNA modifications. Profiling of tRNA methylation and mRNA translation revealed that highly translated mRNAs have higher frequencies of m7G tRNA-decoded codons, and knockdown of METTL1 resulted in decreased translation of mRNAs with higher frequencies of m7G tRNA codons, suggesting that tRNA modifications and codon usage play an essential function in mRNA translation regulation. Our data uncovered novel insights on mRNA translation regulation through tRNA modifications and the corresponding mRNA codon compositions in lung cancer, providing a new molecular basis underlying lung cancer progression. Mis-regulated epigenetic modifications in RNAs are associated with human cancers. The transfer RNAs (tRNAs) are the most heavily modified RNA species in cells; however, little is known about the functions of tRNA modifications in cancers. In this study, we uncovered that the expression levels of tRNA N7-methylguanosine (m7G) methyltransferase complex components methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) are significantly elevated in human lung cancer samples and negatively associated with patient prognosis. Impaired m7G tRNA modification upon METTL1/WDR4 depletion resulted in decreased cell proliferation, colony formation, cell invasion, and impaired tumorigenic capacities of lung cancer cells in vitro and in vivo. Moreover, gain-of-function and mutagenesis experiments revealed that METTL1 promoted lung cancer growth and invasion through regulation of m7G tRNA modifications. Profiling of tRNA methylation and mRNA translation revealed that highly translated mRNAs have higher frequencies of m7G tRNA-decoded codons, and knockdown of METTL1 resulted in decreased translation of mRNAs with higher frequencies of m7G tRNA codons, suggesting that tRNA modifications and codon usage play an essential function in mRNA translation regulation. Our data uncovered novel insights on mRNA translation regulation through tRNA modifications and the corresponding mRNA codon compositions in lung cancer, providing a new molecular basis underlying lung cancer progression. IntroductionLung cancer is one of the most common cancers and a leading cause of cancer-related death worldwide.1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2018.CA Cancer J. 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Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression.Cell. 2016; 165: 1416-1427Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar Moreover, deregulations of tRNA modification enzymes such as human tRNA methyltransferase 9-like (hTRM9L) and histone H2A dioxygenase ALKB homolog 1 (ALKBH1) caused impaired translation regulation and aberrant cancer progression in vitro and in vivo.13Begley U. Sosa M.S. Avivar-Valderas A. Patil A. Endres L. Estrada Y. Chan C.T. Su D. Dedon P.C. Aguirre-Ghiso J.A. Begley T. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.EMBO Mol. Med. 2013; 5: 366-383Crossref PubMed Scopus (72) Google Scholar,14Liu F. Clark W. Luo G. Wang X. Fu Y. Wei J. Wang X. Hao Z. Dai Q. 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Cell. 2018; 71: 244-255.e5Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Okamoto M. Fujiwara M. Hori M. Okada K. Yazama F. Konishi H. Xiao Y. Qi G. Shimamoto F. Ota T. et al.tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.PLoS Genet. 2014; 10: e1004639Crossref PubMed Scopus (71) Google Scholar METTL1 and WDR4 form a methyltransferase complex that catalyzes m7G modification in tRNAs in eukaryotes.15Alexandrov A. Martzen M.R. Phizicky E.M. Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA.RNA. 2002; 8: 1253-1266Crossref PubMed Scopus (166) Google Scholar, 16Lin S. Liu Q. Lelyveld V.S. Choe J. Szostak J.W. Gregory R.I. Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.Mol. Cell. 2018; 71: 244-255.e5Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 17Okamoto M. Fujiwara M. Hori M. Okada K. Yazama F. Konishi H. Xiao Y. Qi G. Shimamoto F. Ota T. et al.tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.PLoS Genet. 2014; 10: e1004639Crossref PubMed Scopus (71) Google Scholar Disruptions of the METTL1 or METTL1/WDR4 complex are correlated with many diseases including microcephalic primordial dwarfism, Galloway-Mowat syndrome, and tumor cell chemoresistance.17Okamoto M. Fujiwara M. Hori M. Okada K. Yazama F. Konishi H. Xiao Y. Qi G. Shimamoto F. Ota T. et al.tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.PLoS Genet. 2014; 10: e1004639Crossref PubMed Scopus (71) Google Scholar, 18Trimouille A. Lasseaux E. Barat P. Deiller C. Drunat S. Rooryck C. Arveiler B. Lacombe D. Further delineation of the phenotype caused by biallelic variants in the WDR4 gene.Clin. Genet. 2018; 93: 374-377Crossref PubMed Scopus (22) Google Scholar, 19Shaheen R. Abdel-Salam G.M. 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Genet. 2018; 61: 468-472Crossref PubMed Scopus (13) Google Scholar Our previous studies demonstrated that METTL1/WDR4-mediated m7G tRNA methylation is critical for stem cell self-renewal and differentiation, providing molecular evidence underlying abnormal m7G tRNA modification in human developmental diseases.16Lin S. Liu Q. Lelyveld V.S. Choe J. Szostak J.W. Gregory R.I. Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.Mol. Cell. 2018; 71: 244-255.e5Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,22Deng Y. Zhou Z. Ji W. Lin S. Wang M. METTL1-mediated m7G methylation maintains pluripotency in human stem cells and limits mesoderm differentiation and vascular development.Stem Cell Res. Ther. 2020; 11: 306Crossref PubMed Scopus (18) Google Scholar Despite its functional importance in stem cell biology and development, the roles of METTL1 and its mediated m7G tRNA modification in lung cancer remain unknown.In this study, we find that METTL1 and WDR4 are significantly upregulated in lung cancer samples and promote lung cancer progression in vitro and in vivo. Mechanistically, METTL1- and WDR4-mediated tRNA m7G modification regulate the translation of mRNAs with m7G tRNA-decoded codon (m7G codon) usage. Our data uncover a clear link between METTL1-mediated m7G tRNA modification and lung cancer progression, providing new insights for the development of therapeutic strategies for efficient lung cancer treatment.ResultsUpregulated METTL1 and WDR4 expression is associated with poor prognosis of lung cancer patientsTo study the role of METTL1- and WDR4-mediated m7G tRNA modifications in regulation of lung cancer, we first analyzed the expression of METTL1 and WDR4 in lung cancers using the lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) datasets from the Cancer Genome Atlas (TCGA). As shown in Figures 1A−1D, the expression levels of both METTL1 and WDR4 mRNAs are significantly upregulated in lung cancer tissues, compared to normal lung tissues. Interestingly, the expression levels of METTL1 and WDR4 mRNAs are significantly co-related in lung cancers (Figures 1E and 1F), suggesting the functional interplay between METTL1 and WDR4. Moreover, high levels of METTL1 and WDR4 are associated with poor patient survival, indicating potential functions of METTL1 and WDR4 in regulation of lung cancer progression (Figures 1G and 1H). To further confirm these results, we analyzed METTL1 expression by immunohistochemistry (IHC) using a lung cancer tissue array. Based on the staining intensities, samples were divided into two groups: the low METTL1 expression group (immunoreactivity score [IRS] < 3) and the high METTL1 expression group (IRS ≧ 3). The representative cases of different groups were shown in Figure 1I. Our data further confirmed that METTL1 expression is significantly upregulated in lung cancer tissues (Figures 1J and 1K). Western blot analysis also verified that both METTL1 and WDR4 are upregulated in human lung cancer specimens, compared to their peri-tumor normal tissues (Figures 1L−1N). Moreover, the m7G modification levels are correspondingly elevated in lung cancer tissues (Figures 1L and 1O). Overall, these results reveal that METTL1 and WDR4 expressions are significantly upregulated in lung cancer and associated with poor prognosis of lung cancer patients, suggesting the potential oncogenic role of METTL1/WDR4 and their mediated m7G tRNA modification in lung cancer.METTL1 inhibition suppresses lung cancer cell proliferation, migration, and invasion in vitroTo investigate the effect of METTL1 on lung cancer, we first knocked down METTL1 expression in A549 and H1299 cells using lentiviruses expressing short hairpin (sh)GFP (negative control [NC]), shMETTL1-1 (shM1-1), or shMETTL1-2 (shM1-2) (Figures 2A and 2B). Interestingly, we found that knockdown of METTL1 reduced the expression of WDR4 but not other methyltransferases including METTL3 and METTL5 (Figures 2A and 2B; Figure S1A), suggesting that METTL1 stabilizes its cofactor WDR4 in lung cancer cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that knockdown of METTL1 inhibited lung cancer cell proliferation in vitro (Figures 2C and 2D). In addition, both A549 and H1299 cells with depleted METTL1 exhibited reduced colony-formation capacities compared to the control groups (Figures 2E and 2F). Cell-cycle analysis revealed that METTL1 knockdown induced an increased ratio of G0/G1-phase cells and decreased the ratio of G2/M-phase cells in lung cancer cells (Figures 2G and 2H). We further carried out wound-healing and invasion assays to evaluate the effect of METTL1 on lung cancer cell migration and metastasis and found that METTL1 knockdown delayed wound closure and suppressed invasive abilities of both A549 and H1299 lung cancer cells (Figures 2I−2L). Overall, these results reveal that METTL1 is essential for lung cancer cell growth, migration, and invasion abilities in vitro.Figure 2Inhibition of METTL1 impairs lung cancer progression in vitroShow full caption(A and B) Validation of the depleted effect of short hairpin (sh)METTL1 by western blot in A549 and H1299 lung cancer cells. (C and D) Knockdown of METTL1 suppressed proliferation of A549 and H1299 cells. (E and F) METTL1-depleted A549 and H1299 lung cancer cells formed less colonies in colony-formation assays. (G and H) Knockdown of METTL1 induced an increased ratio of G0/G1-phase cells and decreased the ratio of G2/M-phase cells in lung cancer cells. (I−L) Knockdown of METTL1 suppressed cell migration (I and J) and invasion (K and L) in A549 and H1299 cells. Data were presented as mean ± SD. Scale bar, 100 μm. (One-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, #p < 0.05, ##p < 0.01, compared to shGFP.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Knockdown of WDR4 impairs lung cancer cell growth and invasion capacities in vitroWDR4 is an important cofactor of METTL1 and is essential for m7G modification on tRNAs.16Lin S. Liu Q. Lelyveld V.S. Choe J. Szostak J.W. Gregory R.I. Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.Mol. Cell. 2018; 71: 244-255.e5Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar To further study the role of m7G tRNA modifications in lung cancer progression, we infected lung cancer cells with lentiviruses expressing shGFP, shWDR4-1, or shWDR4-2 (Figures 3A and 3B). Our data revealed that depletion of WDR4 decreases METTL1 expression, suggesting that WDR4 is essential for maintaining METTL1 protein level (Figures 3A and 3B), which is consistent with the finding in yeast.23Alexandrov A. Grayhack E.J. Phizicky E.M. tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p.RNA. 2005; 11: 821-830Crossref PubMed Scopus (76) Google Scholar We next subjected these cells to proliferation and colony-formation assays and found that WDR4 knockdown led to decreased proliferation and reduced colony-formation activities in lung cancer cells (Figures 3C−3F). In addition, wound-healing and invasion assays further showed that knockdown of WDR4 inhibited migration and invasion of lung cancer cells (Figures 3G−3J), confirming the oncogenic function of WDR4 in lung cancers. Taken together, these results demonstrated that METTL1 and WDR4 play an important function in regulation of lung cancer growth and invasion in vitro.Figure 3WDR4 knockdown inhibited proliferation and migration in lung cancer cellsShow full caption(A and B) Western blot confirmation of WDR4 knockdown using two independent shRNAs in A549 and H1299 cells. (C and D) Cell proliferation of A549 and H1299 cells was suppressed by WDR4 knockdown. (E and F) Knockdown of WDR4 inhibited colony formation in A549 and H1299 cells. (G and H) Wound-healing assay revealed the inhibitory activity of WDR4 knockdown on cell migration. (I and J) Knockdown of WDR4 inhibited A549 and H1299 cell invasion. Data were presented as mean ± SD. Scale bar, 100 μm. (One-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to the shGFP group.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Knockdown of METTL1/WDR4 inhibits tumor growth in vivoWe next performed xenograft tumor-formation assays to evaluate the functions of METTL1/WDR4 in regulation of lung cancers in vivo. Briefly, METTL1 knockdown and control A549 cells were subcutaneously injected into the right dorsal flank of nude mice, and the tumor volumes were measured weekly. Our results showed that larger subcutaneous visible tumors were observed in the control group, whereas only 3 smaller subcutaneous tumors were found in the shM1-1 group, and no subcutaneous tumor was found in the shM1-2 group (Figure 4A). Xenograft tumor growth curve and tumor weight also revealed that METTL1 knockdown significantly reduces tumor growth in vivo (Figures 4B and 4C). IHC analysis confirmed the reduced expression of METTL1 and WDR4 in xenograft tumors from shMETTL1 (shM1) groups (Figures 4D and 4E). In addition, xenograft tumors from shM1 groups showed lower Ki67 expression level, suggesting that METTL1-knockdown xenograft tumors were less malignant than those of the control group (Figure 4F). To further study the role of tRNA m7G modification on lung cancer progression, we also performed the xenograft tumor-formation assays with WDR4-knockdown A549 cells. Similarly, knockdown of WDR4 significantly suppressed lung cancer progression in the xenograft tumor model, as reflected by the reduced tumor size and tumor weight (Figures 4G−4I). Expression of METTL1 and WDR4 was reduced in xenograft tumors from shWDR4 groups (Figures 4J and 4K). In addition, WDR4-depleted tumors were less proliferative as revealed by reduced expression of Ki67 (Figure 4L). Taken together, our in vitro and in vivo assays supported that METTL1 and WDR4 were crucial in the regulation of lung cancer progression.Figure 4Knockdown of METTL1/WDR4 inhibited tumor growth in vivoShow full caption(A) METTL1-depleted A549 cells formed less and smaller xenograft tumors in in vivo experiments (n = 6). (B and C) Growth curve (B) and final tumor weights (C) of xenograft tumors induced by A549 cells with or without METTL1 depletion. (D−F) Representative IHC staining images and quantitative IHC staining scores of METTL1 (D), WDR4 (E), and Ki67 (F) in xenograft tumors. (G) WDR4-depleted A549 cells formed less and smaller xenograft tumors in in vivo experiments (n = 6). (H and I) Growth curve (H) and final tumor weights (I) of xenograft tumors induced by A549 cells with or without WDR4 depletion. (J−L) Representative IHC staining images and quantitative IHC staining scores of WDR4 (J), METTL1 (K), and Ki67 (L) in xenograft tumors. Data were presented as mean ± SEM. Scale bars, 100 μm and 50 μm. (Two-way ANOVA, one-way ANOVA, Student’s t test, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to the shGFP group.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Overexpression of METTL1/WDR4 promotes lung cancer cell growth and invasion capacitiesIn order to further validate the function METTL1/WDR4-mediated m7G tRNA modification in lung cancer progression, we next performed gain-of-function studies in lung cancer cells. Results showed that overexpression of wild-type METTL1 (M1-WT) significantly promotes the cell proliferation and colony formation of lung cancer cells (Figures 5A−5C). In addition, migration and invasion capacities of lung cancer cells were also enhanced by METTL1 overexpression (Figures 5D and 5E). However, overexpression of the catalytic dead mutant METTL1 (M1-Mut) had little function on lung cancer progression (Figures 5A−5E), suggesting that METTL1’s enzyme activity is essential for its function in promoting lung cancer progression. Moreover, overexpression of WDR4 also promoted cell growth, colony formation, migration, and invasion in lung cancer cells (Figures 5F−5J). Taken together, these results demonstrate that METTL1/WDR4 and their mediated m7G tRNA modification are critical for lung cancer progression.Figure 5Overexpressed METTL1/WDR4 promoted cell growth and invasion in A549 cellsShow full caption(A) Western blot confirmation of METTL1 upregulation in A549 cells. (B) Overexpressed METTL1 promoted cell growth in A549 cells. (C) Overexpression of wild-type METTL1 instead of catalytic mutant METTL1 promoted cell colony formation in A549. (D and E) Overexpressed METTL1 promoted migration (D) and invasion (E) of A549 cells. (F) Western blot confirmation of WDR4 upregulation in A549 cells. (G) Overexpressed WDR4 promoted cell growth in A549 cells. (H) Overexpression of WDR4 promoted cell colony formation in A549. (I and J) Overexpressed WDR4 promoted migration (I) and invasion (J) of A549 cells. Data were presented as mean ± SD. Scale bars, 100 μm (D and I); 200 μm (E and J). (Two-way ANOVA, Student’s t test, ∗∗p < 0.01, ∗∗∗p < 0.001, compared to the negative control [NC] group.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)METTL1 depletion reduces tRNA m7G modification and expressionIn order to unveil the mechanisms underlying how METTL1 regulates lung cancer progression, we first performed a northwestern blot to analyze the m7G modification level and found a notable reduction in tRNA m7G modification in METTL1-depleted or WDR4-depleted cells in lung cancer cells (Figures 6A and 6B; Figure S1B). We next performed tRNA reduction and cleavage sequencing (TRAC-seq) to profile m7G modification in tRNAs in lung cancer cells after METTL1 knockdown. Briefly, total RNAs were extracted, and small RNAs were isolated and subjected to DNA oxidative demethylase AlkB demethylation and then reduced and cleaved by NaBH4/aniline treatment. Following purification, the purified RNAs were used for high-throughput sequencing to identify global tRNA m7G modifications at single-nucleotide resolution. Our TRAC-seq result identified a series of tRNA isoacceptors containing m7G modification in A549 lung cancer cells (Figure 6C). METTL1 depletion decreased m7G modification levels in tRNAs as reflected by reduced cleavage scores (Figures 6D and 6E). Analysis of tRNA expression revealed that METTL1 knockdown decreased the expression of the majority of m7G-modified tRNAs (Figures 6F and 6G). Northern blot analysis confirmed that the METTL1 and WDR4 knockdown reduced the expression of m7G-modified tRNAs ValAAC and ProAGG but not the non-m7G-modifiied tRNA GlnCTG (Figure 6H). On the other hand, overexpression of wild-type METTL1 and WDR4 upregulated tRNA m7G modification and enhanced the expression of m7G-modified tRNAs ValAAC and ProAGG but not the non-m7G tRNA GlnCTG (Figure 6I). Overall, these results revealed that METTL1 and WDR4 are essential for tRNA m7G modification and expression.Figure 6METTL1 depletion reduced tRNA m7G modification, m7G tRNA expression, and oncogenic mRNA translationShow full caption(A and B) m7G modification decreased in A549 lung cancer cells with METTL1 or WDR4 depletion. (C) The list of m7G-modified tRNAs in A549 cells identified by TRAC-seq. (D) Representative images of cleavage scores of indicated tRNAs at m7G-modified sties. Decreased cleavage score was shown in the shMETTL1 group, indicating the reduced m7G tRNA modification level in METTL1-depleted cells. (E) tRNA m7G modification level was decreased in METTL1-depleted A549 cells. (F) m7G-modified tRNAs expression levels were decreased in METTL1-depleted A549 cells compared to that of the non-m7G-modified tRNAs. (G) Relative expression profile of m7G-modified tRNAs based on TRAC-seq. The relative expression of each tRNA type was calculated from the combined expression of all of the tRNA genes belonging to the same tRNA type. (H) Northern blotting analyzed the expression levels of representative m7G-modified tRNAs and non-m7G-modified tRNAs in lung cancer cells upon METTL1 or WDR4 depletion. U6 snRNA was used as a loading control. (I) Overexpression of METTL1 or WDR4 increased the expression level of m7G modification and m7G-modified tRNAs in lung cancer cells. (J) Polysome profiling of A549 cells with or without METTL1 knockdown. (K) Translation efficiency (TE) of METTL1-depleted A549 cells was decreased by analyzing the puromycin intake in cells. Overexpression of wild-type METTL1 instead of catalytic inactive mutant METTL1 rescued translation deficiency of METTL1-depleted A549 cells. (L) Scatterplot of gene TE in A549 cells with or without METTL1 depletion. TE was calculated by dividing the RNC-mRNA sequencing signals to input mRNA sequencing signals. (M) Frequency of the m7G-related codons in TE up, TE down, and other genes (others) in METTL1 depletion cells. (N) m7G codon frequency of genes with the top 25% TE was significantly higher than that of the genes with the bottom 25% TE in the NC group. (O) TEs of the identified TE down gene were significantly higher than that of the other and TE up genes in NC cells. (P) Gene ontology analysis of function enrichment in the biological process with TE down genes in METTL1 depletion cells. (Q) Pathway analysis with TE down genes in METTL1 depletion cells. (R and S) qRT-PCR and western blotting confirmed the TEs changes of the identified cell-cycle-related genes. (T) IHC confirmed the decreased expression of the identified TE down gene CCND3 in METTL1-depleted xenograft tumors sections. Data were presented as mean ± SD. Scale bars, 100 μm and 50 μm. (One-way ANOVA, Student’s t test, ∗p < 0.05, ∗∗∗p < 0.001, compared to the shGFP group.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)METTL1 depletion inhibits mRNA translationGiven that tRNAs are essential factors for mRNA translation, we further determined whether m7G tRNA modification was involved in the regulation of mRNA translation in lung cancer cells. Polysome profiling revealed that METTL1 knockdown results in reduction of polysome factions (Figure 6J), suggesting th