m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-β-induced epithelial–mesenchymal transition of lung cancer cells

Abstract

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta–induced epithelial–mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3–mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3′UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta–induced EMT of lung cancer cells. N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta–induced epithelial–mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3–mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3′UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta–induced EMT of lung cancer cells. Epithelial–mesenchymal transition (EMT) is one of the crucial mechanisms causing cancer malignancies, such as invasion, metastasis, and resistance to therapy (1Dongre A. Weinberg R.A. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer.Nat. Rev. Mol. Cell Biol. 2019; 20: 69-84Crossref PubMed Scopus (1419) Google Scholar, 2Pastushenko I. Blanpain C. EMT transition states during tumor progression and metastasis.Trends Cell Biol. 2019; 29: 212-226Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar). During EMT, tumor cells lose epithelial characters such as cell polarity and cell contacts and acquire invasive stem cell–like properties that expand their ability for local invasion and metastasis. A major inducer of EMT is transforming growth factor-beta (TGF-β) along with the cytokines and growth factors secreted by the tumor microenvironment. EMT is characterized by the dynamic and reversible changes in epithelial and mesenchymal gene expression (3Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (5130) Google Scholar). Epithelial cell markers such as E-cadherin and claudins are downregulated, whereas mesenchymal markers including vimentin, fibronectin, and N-cadherin are upregulated during EMT. Many transcription factors (TFs) are involved in the transcriptional regulation of EMT-related genes. Especially, TFs such as ZEB family, SNAIL family, and TWIST can activate EMT through the transcriptional repression of E-cadherin. Recently, nonredundant functions of EMT-related TFs have been emphasized (4Stemmler M.P. Eccles R.L. Brabletz S. Brabletz T. Non-redundant functions of EMT transcription factors.Nat. Cell Biol. 2019; 21: 102-112Crossref PubMed Scopus (244) Google Scholar). Because of the differential expression patterns in different tumor types, EMT-related TFs would have different functions and different target genes in a context-dependent manner. Therefore, careful analyses and discussions are important to understand their roles in various cancers. In addition, epigenetic regulations are considered as one of the critical mechanisms for EMT owing to its phenotypic plasticity (5Tam W.L. Weinberg R.A. The epigenetics of epithelial-mesenchymal plasticity in cancer.Nat. Med. 2013; 19: 1438-1449Crossref PubMed Scopus (875) Google Scholar, 6Serrano-Gomez S.J. Maziveyi of epithelial-mesenchymal transition through epigenetic and 15: PubMed Google Scholar). we have investigated and found the roles of and in the transcriptional regulatory network during EMT S. A. T. epithelial-mesenchymal transition of cancer cells by 2014; PubMed Scopus Google Scholar, S. A. T. to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. T. to epigenetic progression of the epithelial-mesenchymal transition of lung and cancer Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. T. T. is involved in the epigenetic regulation of epithelial-mesenchymal transition in lung and cancer cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). N6-methyladenosine (m6A) is the most internal chemical modification of mRNAs and in and is in mRNA and translation S. and mRNA Rev. Mol. Cell Biol. 2019; 20: PubMed Scopus Google Scholar). modification is controlled by a of proteins identified as and 3 and the m6A to m6A the and function as to the of m6A which a dynamic of this modification J. C. and context-dependent functions of and 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). the family, family, and to the modification is that m6A modification an important role in including and C. A mechanisms to J. Scopus Google Scholar). have that m6A regulation is involved in the and progression of various of cancer J. S. T. The critical role of in 2019; PubMed Scopus Google Scholar, J. modification in and roles and implications in Full Text Full Text PDF PubMed Scopus Google Scholar). been that JUN of and are by various and are involved in and in cell and 20: PubMed Scopus Google Scholar). In with to EMT that of JUN E-cadherin expression and N-cadherin in cells S. of cell invasion, and EMT in cell PubMed Scopus Google and expression in cancer cells cancer cell by PubMed Scopus Google Scholar). During transforming growth factor-beta EMT in epithelial cell JUNB the of factors such as and which are for and A. S. to repression and the epithelial-mesenchymal transition in to transforming growth Cell Biol. PubMed Scopus Google Scholar). to be involved in EMT of and cell through the of S. A. S. of and transition by with PubMed Scopus Google Scholar). These that the of JUN in EMT and the target genes be different in various cancer we have that JUNB is one of the important TFs by m6A during EMT of A549 and LC2/ad lung cancer cell S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google Scholar). the stability of JUNB mRNA through m6A the function of JUN and its regulation by m6A modification during EMT in lung cancer cells are In this study, we to the function and regulatory of JUN TFs in the expression of epithelial and mesenchymal genes during EMT of lung cancer that JUN and JUNB but not to the EMT-inducing gene expression program by regulating different mesenchymal genes. We found that m6A modification by the expression of JUN the protein but controlled JUNB expression the during EMT, which by the different m6A reader the of JUN TFs in EMT of lung cancer we the changes in expression of and by A and and that the expressions of JUN and JUNB but not of mRNA were by in A549 and LC2/ad lung cancer cell We of JUN and JUNB but not of protein in A549 and LC2/ad cells by These results the of JUN and JUNB TFs in EMT we the of JUN in A549 and LC2/ad cells to their functions in EMT We different and for gene and that the the expression of the target gene in cells by During EMT, epithelial cells acquire a mesenchymal by an epithelial cell and mesenchymal cell markers such as and (3Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (5130) Google Scholar). We the expression of by and The for gene in the expression of EMT-related genes in cells and and the of in A549 cells were as the results in the of JUN and JUNB the expression of an epithelial in the of and its transcriptional repression by in A549 and LC2/ad cells A and result the important of JUN and JUNB TFs to the transcriptional regulation during EMT of lung cancer cells. the mesenchymal JUN and JUNB the expression of gene but of its expression in cells A and of JUNB but not of JUN its expression in the or the of in A549 cells and expression to be in LC2/ad cells. the of JUN but not of JUNB the expression of in cells and the different functions of JUN and JUNB TFs in the regulation of mesenchymal genes. In of not the expression of these EMT-related genes and of to EMT of lung cancer cells. We these of JUN or JUNB the protein expression of vimentin, fibronectin, and N-cadherin during EMT of A549 and LC2/ad cells A and of JUN or JUNB protein E-cadherin expression and its We that JUNB the of whereas JUN the N-cadherin by and These results that JUN and JUNB TFs controlled the expression of common or specific EMT-related target genes and played a crucial role in EMT of A549 and LC2/ad lung cancer cell we have that JUNB is one of the important TFs by m6A during EMT process S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google Scholar). to JUN controlled by in A549 and LC2/ad cells. We the m6A of JUN mRNA by m6A The JUN mRNA by in A549 and LC2/ad lung cancer cells and the m6A of JUN mRNA in the of and the the of m6A and been for EMT S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google m6A of JUN mRNA to an important regulatory role in EMT. We found that the protein of JUN in cells and the of JUN protein and JUN mRNA expression in the of and and Therefore, the of JUN protein by not be by the mRNA expression These results that m6A the expression of JUN protein but not of JUN mRNA. is in contrast with the of m6A-dependent regulation of a in JUNB mRNA by S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google Scholar). we that the of JUN protein in the cells be to the in protein stability or the stability of JUN the and cells were with to protein the in A549 and LC2/ad cells and of the that the expression of JUN protein in the and cells and that protein stability of JUN not by m6A These results that the m6A-mediated expression of JUN protein to the regulation of translation process the mRNA regulation and protein stability. In the study, we that the stability of JUNB mRNA through m6A by during EMT process S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google Scholar). the we to which of JUNB responsible for the m6A-dependent We a by or 3′UTR of JUNB and an internal These were into the and A549 and and the expression of mRNA were the of the JUNB 3′UTR in the cells in the cells a of mRNA for the with JUNB 3′UTR These results that 3′UTR of JUNB the responsible for mRNA regulation by to the of m6A J. and of the with implications for the Scopus Google are m6A motifs within 3′UTR of JUNB in A549 cells We the of to the these m6A and m6A for the and A549 cells with or that and a of m6A in the of The and but not a of m6A in the cells result that the and of JUNB 3′UTR the m6A controlled by are m6A within the and we the for m6A and and the These were into the and A549 and and mRNA expression were The and of m6A in a of and mRNA expression in the and the and the in and mRNA, and the not with and the of and mRNA by the and not in the cells and the of in the regulation through the m6A These results that the and of JUNB 3′UTR were the target of for the regulation of JUNB mRNA. we the results the in a different We JUNB expression or and into the and A549 cells. The to the and that the JUNB a of JUNB protein and mRNA in the the of 3′UTR in we JUNB expression for and the m6A and in the We that the and a of JUNB protein and mRNA, but the in the cells and this m6A regulation of JUNB expression not in the cells and we that the and of JUNB 3′UTR were responsible for m6A-mediated regulation of JUNB mRNA in A549 lung cancer cells. We to which of JUN mRNA involved in the regulation of protein expression by We or 3′UTR of JUN and the in the and A549 cells. we not we JUN expression or to the expression the and cells. in the and but not expression of JUN protein in the cells. of JUN mRNA for the result with the that JUN expression controlled by the protein These results that 3′UTR of JUN the critical for the regulation by The of m6A J. and of the with implications for the Scopus Google m6A motifs within 3′UTR of JUN in A549 cells We the to to these m6A and m6A for the cells that the and but not a of m6A by and a of m6A in the cells that these the target m6A We found m6A within the and and JUN expression for m6A and and the that of and in an of JUN protein in the cells The of or but the not by or We that these m6A not in the expression of JUN mRNA The m6A regulation of JUN protein not in the cells and the of in the regulation through the m6A we that the and of JUN 3′UTR were responsible for regulation of JUN protein expression in A549 lung cancer cells. results that controlled the expression of JUNB the mRNA but controlled JUN expression the protein We that different m6A reader proteins be involved in the m6A-mediated regulation of JUN and JUNB. the m6A reader proteins for JUN and we the of of m6A reader the expression of JUN and JUNB proteins in A549 lung cancer cells of m6A and by the by and A and We to JUN and JUNB proteins in m6A reader cells and the with in the and cells. The results that JUN protein in and JUNB protein with We to and as the m6A reader proteins for JUN and the of and in the regulation of JUN and JUNB we the in protein and mRNA expression in A549 and LC2/ad lung cancer cells with or We found that the protein of JUN in A549 and LC2/ad cells and the of JUN JUN mRNA expression in the or the of and In the expression and of JUNB protein and mRNA in A549 and and LC2/ad and cells. These results are with the results for the m6A-dependent regulation of JUN protein and JUNB mRNA. the the m6A reader proteins and JUN or JUNB mRNA, we The or A549 cells were with and the cell were in the of with or and the by with The of protein and JUN mRNA in the cells with but this in the cells were and We the and JUNB mRNA, which by These results the of mRNA and mRNA in lung cancer cells and that these were the m6A by we JUN expression for and the m6A and into the cells. We not the of JUN protein in the cells and JUNB that the m6A regulation of JUNB mRNA not in the cells and These results the of and in the m6A-mediated regulation of JUN and Therefore, that and were the most m6A reader proteins involved in the regulation of expression of JUN protein and JUNB mRNA, In this study, we discovered the m6A regulation of JUN and JUNB which an and nonredundant role in gene expression program during EMT of A549 and LC2/ad lung cancer cells. that the translation process of JUN protein and the stability of JUNB mRNA through the m6A motifs 3′UTR of JUN and JUNB. In addition, and were identified as the most m6A reader proteins for JUN and We that the different modes of m6A-mediated regulation of JUN and JUNB TFs during EMT process be to the functions of these m6A reader JUN TFs the expression of target genes and in the regulation of and in cell and 20: PubMed Scopus Google Scholar). that of JUN to EMT process in different of cancer cell and invasion S. of cell invasion, and EMT in cell PubMed Scopus Google Scholar, cancer cell by PubMed Scopus Google Scholar, A. S. to repression and the epithelial-mesenchymal transition in to transforming growth Cell Biol. PubMed Scopus Google Scholar, S. A. S. of and transition by with PubMed Scopus Google Scholar). the target genes controlled by JUN and the modes of regulation are the mechanisms during EMT are to be In this study, we found that of JUN and JUNB the expression of epithelial and mesenchymal genes during EMT of A549 and LC2/ad lung cancer cells and the EMT-related genes as as we the of the expression of JUN and JUNB by and that JUN and JUNB TFs controlled the expression of different mesenchymal and and is with the gene regulatory of JUN and JUNB by the S. of cell invasion, and EMT in cell PubMed Scopus Google Scholar, A. S. to repression and the epithelial-mesenchymal transition in to transforming growth Cell Biol. PubMed Scopus Google Scholar). results that common and functions of JUN and JUNB TFs were important in the transcriptional regulation during EMT of lung cancer cells. is with the that EMT-related TFs have and functions in EMT which can be specific to the cancer (4Stemmler M.P. Eccles R.L. Brabletz S. Brabletz T. Non-redundant functions of EMT transcription factors.Nat. Cell Biol. 2019; 21: 102-112Crossref PubMed Scopus (244) Google Scholar). we the gene regulatory network in EMT controlled by the different EMT-related their roles in EMT, a of the and be by such as for and in that m6A modification by in many pathological processes including cancer C. A mechanisms to J. Scopus Google Scholar, J. S. T. The critical role of in 2019; PubMed Scopus Google Scholar, J. modification in and roles and implications in Full Text Full Text PDF PubMed Scopus Google Scholar). the important function of and its in various cancer mRNA in stem cell and tumor progression A. A. A. role of modification in cells and PubMed Scopus Google Scholar, T. tumor progression an in 2019; PubMed Scopus Google in progression J. J. The m6A progression by regulating the m6A of 2019; PubMed Scopus Google in cancer cancer progression 2019; PubMed Scopus Google and in cancer J. J. of cancer progression by PubMed Scopus Google Scholar). Especially, with to EMT the important roles of m6A regulation in and cells J. J. the epithelial mesenchymal transition of cancer cells and translation of 2019; PubMed Scopus Google and in cancer cells T. S. J. Xu N6-methyladenosine to the and of Cell PubMed Scopus Google Scholar, C. R. N6-methyladenosine modification is critical for epithelial-mesenchymal transition and of 2019; PubMed Scopus Google in cancer cells J. growth and invasion through the regulation of translation and epithelial to mesenchymal Full Text Full Text PDF PubMed Scopus Google and in epithelial cells C. T. J. J. modification of mRNA is involved in the EMT in cancer of the Full Text Full Text PDF PubMed Scopus Google Scholar). We have that the m6A regulation of JUN and JUNB by a critical role in the expression of the EMT-inducing gene expression program of A549 and LC2/ad lung cancer cells. the phenotypic of EMT by S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google be by the expression of JUN and JUNB for EMT-related TFs and factors controlled by are to the for the function in EMT of lung In addition, the of m6A modification are controlled by m6A and we the expression of and m6A in EMT of A549 and LC2/ad cells and were in the of JUN A and The expression of and mRNAs and proteins were not in to in cells We a in their expressions but the were not In the S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google we a in the expression of during EMT of A549 and LC2/ad cells. These results that m6A to the m6A modification of mRNAs in EMT of lung cancer cells. the responsible of JUNB and JUN for the m6A regulation by we and expression different of We JUNB 3′UTR as the m6A regulatory in and JUN 3′UTR as the responsible in the expression We not the for the in the of we that the of JUN be for the protein regulation by m6A we have identified the specific m6A for JUN and which are associated with the m6A-dependent regulation of JUN protein and JUNB mRNA 3 and these results are the the m6A motifs in the J. and of the with implications for the Scopus Google we the that m6A be the for or m6A the of these m6A in the regulation of JUN and JUNB. In this study, we and as the m6A reader proteins for JUN and the the most the expression m6A reader proteins have been in we the that m6A reader proteins be and m6A to that and were the most m6A reader proteins for JUN and to protein and which been as to the mRNAs and their and translation N6-methyladenosine proteins in Scholar). have its function to translation m6A modification N6-methyladenosine proteins in Scholar). These result that is the m6A reader for m6A-dependent expression of JUN protein to be associated with the regulation of translation process and with and functions through with translation and of PubMed Scopus Google Scholar). We of JUN protein expression in the cells which the of to the m6A regulation of the of been that with the and A. reader mRNA PubMed Scopus Google and the translation translation and of PubMed Scopus Google Scholar). Therefore, would be the important to the of regulation of JUN protein expression during EMT. Recently, been to be associated with the progression of of translation of to and of cancer properties in S. R. protein of cancer cells in through PubMed Scopus Google and cancer through m6A-dependent and growth expressions S. the translation of gene to cancer Full Text Full Text PDF PubMed Scopus Google Scholar). in cancer involved in EMT, cell and invasion cancer progression and by mRNA in an Med. PubMed Google Scholar). The expression of E-cadherin whereas N-cadherin and expressions were through the mRNA by cancer progression and by mRNA in an Med. PubMed Google Scholar). These that the function of and the target mRNAs have not been is important to the the common or function of in the progression of cancer including EMT. is a of which m6A and mRNA is with results that the m6A-dependent expression of JUNB mRNA. is as an which is in and various of but is in growth protein in PubMed Scopus Google Scholar). The functions of in have been as an m6A reader protein growth protein in PubMed Scopus Google Scholar). Recently, been that cell and invasion of cells and cancer cells by the target mRNAs including of by proteins mRNA stability and Cell Biol. 20: PubMed Scopus Google and controlled stem cell of cancer cells by the of mRNA S. J. T. transcription in cancer in a and 2019; PubMed Scopus Google Scholar). to have been m6A regulation by in EMT A that EMT by mRNA in and but the m6A A. S. mesenchymal cell properties and of cells by the expression of and 2013; PubMed Scopus Google Scholar). is to be involved in the transcription of a mesenchymal A. S. mesenchymal cell properties and of cells by the expression of and 2013; PubMed Scopus Google which is to the of JUNB in this Therefore, would be to the role of in EMT by its function in the transcriptional regulation of a of mesenchymal genes including In we the different modes of m6A regulation for JUN and JUNB which an role in the EMT-inducing gene regulation during EMT of lung cancer cell m6A reader and were to with JUN and JUNB mRNA, and to the m6A-dependent regulation of JUN protein translation and JUNB mRNA stability. plasticity of cancer cells by EMT to progression through and EMT process the to cancer and the of in EMT is for We that m6A-dependent regulation of EMT-related TFs a target in and therapy the were as A. the expression of and the invasive of the J. PubMed Scopus Google Scholar). The were and into The for were S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google and are in We different and for that have for of gene were and and JUN family, the have EMT the gene expression and the of one for gene were in the and the of JUN and JUNB the in were the and in for The were into T. or A. S. T. functions as a of during PubMed Scopus Google to and we the the and The were with as A. S. T. functions as a of during PubMed Scopus Google and into to the the and 3′UTR of JUN or JUNB the were by and into and of the expression for m6A we to the for the m6A into 3′UTR of JUN JUN m6A in The were by the different The JUNB m6A were by the JUNB m6A The of the were by The were for and lung cancer cell lines, A549 and were and and in and the of and with S. A. T. to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). These cell are a for EMT and changes in cell and EMT-related gene expression by of of for The for the and of and or the of expression were the as A. the expression of and the invasive of the J. PubMed Scopus Google Scholar, S. A. S. epithelial-mesenchymal transition of cancer cells by regulating the expression of the 2013; PubMed Scopus Google Scholar). with a and to as S. A. S. epithelial-mesenchymal transition of cancer cells by regulating the expression of the 2013; PubMed Scopus Google Scholar). were with The are with the were the and the for the were A. the expression of and the invasive of the J. PubMed Scopus Google Scholar, S. A. S. epithelial-mesenchymal transition of cancer cells by regulating the expression of the 2013; PubMed Scopus Google Scholar, S. A. T. is involved in transforming growth epithelial-mesenchymal transition of lung and cancer cell 2014; PubMed Scopus Google and are in cells were in as A. S. T. to epigenetic progression of the epithelial-mesenchymal transition of lung and cancer Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The were and to The in this Cell Cell and a to that the of protein the protein in the with we the of in a protein stability the cells were with to protein the cell were for m6A to the S. A. T. m6A to epithelial-mesenchymal transition of lung cancer cells through the regulation of PubMed Scopus Google Scholar). of with in of and with and the were with The were with by the and were by A549 cells with or were in in and the or 3′UTR of JUNB The of and were with the to the The for the mRNA expression of and the for and to the S. A. T. to the epigenetic regulation of epithelial-mesenchymal transition in lung cancer cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells were with for and the were by The cell were with with and The were with or to The were with and were by input to the of the the not are within the and The and in this are the The that have of with the of this We for We and T. of and for T. S. S. and S. R. T. and T. T. S. R. A. and T. T. S. and T. T. S. T. and A. I. T. S. in by for to T. to and to A. the of and of

Topics & Concepts