A snoRNA–tRNA modification network governs codon-biased cellular states [RNAseq & Riboseq]

The dynamic balance between tRNA supply and codon usage demand is a fundamental principle in the cellular translation economy. However, the regulation and functional consequences of this balance remain unclear. Here, we use PARIS2 interactome capture, structure modeling, conservation analysis, RNA–protein interaction analysis, and modification mapping to reveal the targets of hundreds of snoRNAs, many of which were previously considered orphans. We identify a snoRNA–tRNA interaction network that is required for global tRNA modifications, including 2'-O-methylation and others. Loss of Fibrillarin, the snoRNA-guided 2'-O-methyltransferase, induces global upregulation of tRNA fragments, a large group of regulatory RNAs. In particular, the snoRNAs D97/D133 guide the 2'-O-methylation of multiple tRNAs, especially for the amino acid methionine (Met), a protein-intrinsic antioxidant. Loss of D97/D133 snoRNAs in human HEK293 cells reduced target tRNA levels and induced codon adaptation of the transcriptome and translatome. Both D97/D133 single and double knockouts in HEK293 cells suppress Met-enriched proliferation-related gene expression programs, including, translation, splicing, and mitochondrial energy metabolism, and promote Met-depleted programs related to development, differentiation, and morphogenesis. In a mouse embryonic stem cell model of development, knockdown and knockout of D97/D133 promote differentiation to mesoderm and endoderm fates, such as cardiomyocytes, without compromising pluripotency, consistent with the enhanced development-related gene expression programs in human cells. This work solves a decade-old mystery about orphan snoRNAs and reveals a function of snoRNAs in controlling the codon-biased dichotomous cellular states of proliferation and development. Overall design: To investigate the novel functions of snoRNAs that guide 2'-O-methylation (Nm) modifications on tRNAs, we established SNORD97, SNORD133 single and double knock out HEK293 and mESCs using CRISPR/Cas9 technology, we also generated FBL and DKC1 knock down HEK293 cells by siRNAs. Total RNA sequencing was performed to analyze comparative gene expression profiles. Small RNA sequencing was performed to analyze the tRNA fragmentation after loss of the snoRNA-guided 2'-O-methylation modifications on tRNA. Ribosome profiling sequencing (ribo-seq) was performed to analyze the cause of reduced translation after CRISPR knockout of the D97/D133 family of snoRNAs,