28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures.
The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore.
Franck C
FC
Courtes
Department of Biological Engineering and Center for Environmental Health Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Chen
C
Gu
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; AbbVie Pte. Ltd., 8 Biomedical Grove, #03-01 Neuros, Singapore 138665, Singapore.
Niki S C
NS
Wong
Department of Biological Engineering and Center for Environmental Health Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Peter C
PC
Dedon
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore.
Miranda G S
MG
Yap
Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. Electronic address: cheld@nus.edu.sg.
Dong-Yup
DY
Lee
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D011560:Pseudouridine / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000235:genetics*; D012270:Ribosomes / Q000187:drug effects / Q000502:physiology; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
D012339:RNA, Ribosomal, 28S; D020537:RNA, Small Nucleolar; D011560:Pseudouridine; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus
CHO cells culture; Inducible pseudouridylation; Translatome; mTOR pathway; snoRNA U19