(a) Western blot of proteins from LARP4 codon swap (CS) constructs. WT = wild type LARP4; others are described in the text. (b) Northern blot of RNA from same cells in a. (c) Cellular tRNA index (ctAI) scores of the CRD regions of CS constructs. ctAI scores plotted on x-axis and Flag-LARP4 mRNA levels relative to Flag-LARP4-WT on the y-axis; N = 7 biological replicates for CS-R and CSb, N = 5 biological replicates for CS-W, CS-I, and CS-B, N = 4 biological replicates for CSc, N = 3 biological replicates for CS-Tyr and CSa; error bars reflect the s.e.m. (d) Top: Sliding window ctAI-based score plot depiction of the full length LARP4 ORF of 724 codons (numbered on X-axis). Regions scoring below 0.4 (Y-axis) are colored red (see text). The position of the CRD codons 287–358 are indicated by the bracket. Bottom: The CRD codons are shown with single letter amino acids underneath and with numbers above that correspond to their cognate tRNAs as follows. Numbers 1–11 designate the rank order low level tRNAs in Bin-1 from Table 1; red = wobble decoded, blue = Watson:Crick decoded. Symbols γ and λ designate weak (all-A + T) codons that must be wobble decoded (see text) by non-Bin-1 tRNAs; note that #11 (F, Phe) is also a weak (all-A + T) codon. (e) Northern blot 48 hr after tRNA gene-containing plasmids were transfected into HEK293 cells. (f) Western blot of extracts from HEK293 cells transfected with empty pUC19 or plasmids containing tRNA genes for PheGAA, ProAGG, ThrUGU or combinations as indicated above the lanes, together with empty pCMV2 or F-LARP4-WT, and GFP. Antibodies used are indicated to the left of the panels. (g–j) HEK293 cells transfected with empty plasmid (ep) or 2, 4, or 6 ug of tRNAThrUGU plasmid together with empty pCMV2 (empty vector), F-LARP4-WT, CS-W, CS-I, or CS-B, and GFP. (g) Western blots as indicated. (h) Quantification of LARP4 in g as indicated. (i) Northern blot. (j) quantification of LARP4 mRNA/Rpl32 mRNA from northern blot in i.