Selective gene expression maintains human tRNA anticodon pools during differentiation

Transfer RNAs are essential for translating genetic information into proteins. The human genome contains hundreds of predicted tRNA genes, many in multiple copies. How their expression is regulated to control tRNA repertoires is unknown. Here we combined quantitative tRNA profiling and chromatin immunoprecipitation with sequencing to measure tRNA expression following the differentiation of human induced pluripotent stem cells into neuronal and cardiac cells. We find that tRNA transcript levels vary substantially, whereas tRNA anticodon pools, which govern decoding rates, are more stable among cell types. Mechanistically, RNA polymerase III transcribes a wide range of tRNA genes in human induced pluripotent stem cells but on differentiation becomes constrained to a subset we define as housekeeping tRNAs. This shift is mediated by decreased mTORC1 signalling, which activates the RNA polymerase III repressor MAF1. Our data explain how tRNA anticodon pools are buffered to maintain decoding speed across cell types and reveal that mTORC1 drives selective tRNA expression during differentiation.


Replication
For tRNA-seq, ATAC-seq, RNA-seq and ChIP-seq experiments, 2 biological replicates (independent differentiations) were performed, and correlation analysis were conducted to ensure the consistency between replicates.For H3K27me3 ChIP-seq, H3K9me3 ChIP-Seq, and H3K4me3 ChIP-Seq in NPC, a single replicate per cell type were performed.For ribosome profiling where cycloheximide (CHX) and tigecycline (TIG) were present in lysis buffer, 2 biological replicates for hiPSC and NPC were performed, while only one replicate was performed for the CHX-only sample.For CRISPRi and following functional studies, 2 clones were selected for each sgRNA.All attempts of replications were successful.
Randomization No randomization was performed.This study was carried out in the kucg_2 hiPSC line and its differentiated counterparts, as well as in wibj_2 hiPSC cells and HEK293T/17.Covariates control is not applicable due to the small number of cell lines used.

Blinding
The investigators were not blinded to the group as no human subjects or clinical samples were involved and no subjective measurements were taken.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.antibody-c-10?productCanUrl=oct-3-4-antibody-c-10&_requestid=526329) Anti-SOX2 E-4 (Santa Cruz, #sc-365823): according to the manufacturer, this mouse monoclonal antibody is specific for an epitope mapping between amino acids 170-201 within an internal region of Sox-2 of human origin and is recommended for is recommended for detection of Sox-2 of mouse, rat and human origin by immunofluorescence; cited in >260 publications (https://www.scbt.com/p/sox-2-antibody-e-4?requestFrom=search).
Anti-POLR3A/RPC1 (Cell Signaling Technology, #12825): according to the manufacturer, this monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val613 of human POLR3A protein and has been validated for use in ChIP-Seq (https://www.cellsignal.com/products/primary-antibodies/polr3a-d5y2d-rabbit-mab/12825).Additional validation we performed included Western blotting (single band at the expected MW of POLR3A/RPC1) and extensive characterization of ChIP-Seq datasets (presence of clearly defined strong peaks at Pol III target genes and absence of ChIP signal from other genomic regions).
Anti-POLR3G/RPC7α (Santa Cruz, #sc21754): according to the manufacturer, this monoclonal antibody raised against recombinant human RPC32/POLR3G/RPC7α subunit of RNA polymerase III and cited in >6 publications (https://www.scbt.com/p/pol-iii-rpc32antibody-c32-1).Additional validation we performed included Western blotting (single band at the expected MW of POLR3G and its substantial decrease upon POLR3G knockdown by CRISPRi) and extensive characterization of ChIP-Seq datasets (presence of clearly defined strong peaks at Pol III target genes and absence of ChIP signal from other genomic regions).
Anti-BRF1 (Abcam, #ab264191): according to the manufacturer, this polyclonal antibody was raised against a synthetic peptide within human BRF1 aa 627-677 (https://www.abcam.com/products/primary-antibodies/brf1-antibody-ab264191.html).Additional validation we performed included Western blotting (single band at the expected MW of BRF1 and its substantial decrease upon BRF1 knockdown by CRISPRi) and extensive characterization of ChIP-Seq datasets (presence of clearly defined strong peaks at Pol III target genes and absence of ChIP signal from other genomic regions, inlcuding RNU6-1, at which Pol III is assembled via BRF2).

April 2023
Anti-MAF1 (Santa Cruz; #sc-515614 X): according to the manufacturer, this monolconal antibody specific for an epitope mapping between amino acids 99-122 within an internal region of MAF1 of human origin; cited in 3 publications (https://www.scbt.com/p/maf1-antibody-h-2).Additional validation we performed included Western blotting (single band or smear at the expected MW of MAF1 depending on phosphorylation status and its substantial decrease upon MAF1 knockdown by CRISPRi).
nature portfolio | reporting summaryApril 2023 Data exclusions No data was excluded.