Fig 2: Depletion of U16 snoRNA by ASO treatment does not impair the expression of the host gene. (A) Schematic depiction of the U16 snoRNA gene locus. The partial host pre-mRNA (RPL4) is shown. The gray boxes indicate intronic snoRNAs (U16 and three isoforms of U18 snoRNA) in the host gene. E2–E7, exons of RPL4 pre-mRNA. (B) ASOs targeting U16 did not affect the levels of U18 snoRNAs encoded in the same host pre-mRNA. HeLa cells were treated for 48 h with different concentrations of ASO462026. Total RNA was prepared and subjected to northern hybridization with probes specific to U16 and U18 snoRNAs. U4 snRNA was detected as a loading control. (C) HeLa cells were treated for 48 h with 50 nM of three different ASOs targeting U16 snoRNA. Northern hybridization was performed as in (B). Ethidium bromide staining of 5 S rRNA is shown in lower panel. (D) The mRNA level of the host gene (RPL4) was not changed by U16 ASOs. Aliquots of RNAs as in (C) were separated in a 6% polyacrylamide gel, and RPL4 mRNA was detected by northern hybridization. U3 snoRNA served as a loading control. (E) Depletion of U16 by ASO did not alter the protein level of RPL4. HeLa cells were treated with U16 ASO (462026) for different times, as indicated above lanes. Whole cell extracts were prepared and subjected to western analysis using an anti-RPL4 antibody (left panel). As a control for loading, the same membrane was re-probed with an alpha-tubulin antibody. In addition, total RNA was prepared from an aliquot of test cells and subjected to northern hybridization, using probes specific to U16. The same membrane was re-probed for U80 snoRNA, which served as a loading control (right panel).

Fig 3: METTL5 affects cell proliferation and metabolism through differential translation of specific transcripts.A, cell proliferation of HeLa METTL5-WT and METTL5-KO cells over time as measured by CyQuant assay. Means (squares) and SDs (bars) are indicated for four replicate wells per condition. B, polysome profiles from HeLa METTL5-WT and METTL5-KO cells as measured by normalized absorbance over a 5 to 50% sucrose gradient. C, Western blots of polysome fractions from panel B for METTL5, RPL4 (large ribosomal subunit marker), RPS6 (small ribosomal subunit marker), and eIF3A and eIF4E (translation initiation factors). D, effects of METTL5 knockout on translation and transcription as displayed by log2(fold change) of translation efficiency (TE) versus log2(fold change) of reads per kilobase of transcript per million mapped reads (RPKM) from ribosome profiling input and ribosome-protected fragment sequencing of HepG2 cells. Case = METTL5-KO, Control = METTL5-WT. E, volcano plot of the negative log10 of adjusted p-value versus log2(fold change) of normalized gene expression from ribosome profiling of HepG2 METTL5-KO versus METTL5-WT cells. F, biological process gene ontology (GO) functional enrichment analysis for translationally upregulated transcripts in HepG2 METTL5-KO compared with METTL5-WT cells. D and F are modified from output of RiboToolKit (61). All indicated band sizes in Western blots are in kilodaltons (kDa).

Fig 4: Translationally Downregulated Proteins following eIF2a Phosphorylation Are Long-Lived(A) Experimental design (top panel) to measure mitochondrial translation. 35S methionine/cysteine incorporation into newly translated mitochondrial proteins (encoded by mtDNA) following chloramphenicol (Cm; 50 µg/mL) or Tm (2.5 µg/mL) treatment of HEK293T cells for the indicated times. Cytoplasmic translation was inhibited using emetine (100 µg/mL). ATF4 was used as a positive control for the Tm treatment. Representative results of at least three independent experiments are shown.(B) Representative immunoblots showing the abundance of RPL4, RPS6, MRPL44, MRPL28, ATF4, and tubulin proteins at the indicated times after treatment of HeLa cells with 2.5 µg/mL Tm. ATF4 was used as a positive control for the Tm treatment.(C) Same as (B), but following 20 µg/mL cycloheximide (CHX) treatment. POMP (a short-lived protein) was used as a control for the CHX treatment.(D) Histogram showing the distribution of protein half-lives encoded by translationally down- or upregulated mRNAs (Down or Up) at 2 h following Tm injection.(E) Boxplot of half-lives of proteins encoded by the translationally down- or upregulated mRNAs (Down or Up), all 79 CytoRPs, or all 82 MitoRPs at 2 h following Tm injection.(F) Same as (E), but with exclusion of the RPs (Down w/o RPs).In (E) and (F), protein half-lives were analyzed from Sandoval et al. (2013). **p < 0.01 and ***p < 0.001, as determined by an unpaired Wilcoxon test. See also Figures S8 and S9.

Fig 5: Analysis of in vivo ribosome functional states by ribosome profiling Immunoblot of eEF3 depletion over time. Same amount of cells were harvested at indicated time points, lysed, and subjected to immunoblotting using antibodies against eEF3 or PGK1.Growth of WT and eEF3d cells on YPGR and YPD + auxin plates. Plates were incubated at 30°C for 1 day.Representative run-off polysome profiles for WT and eEF3d strains. Polysome-to-monosome ratios (P/M) are normalized to WT ratios. Data are presented as mean ± SD, n = 2.Polysome profiles from WT or eEF3d cells with CHX added during cell lysis to stop translation (top). Fractions were analyzed by immunoblotting using antibodies against eEF1A, eEF2, or RPL4 (bottom).De-enriched peptide motifs associated with ribosome pausing at the E, P, and A sites in the absence of eEF3. Peptide motif logos from two biological replicates are shown.