Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing

Abstract

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.

Keywords: AZIN1; antizyme inhibitor; eIF5A; near cognate; polyamines; ribosome; spermidine.

Figure 1. Sequence element near the C-terminus of the AZIN1 uCC is required for polyamine stimulation of translation initiation at the upstream AUU start codon

(A) HEK293T cells treated with DFMO (polyamine-depleted) or DFMO+SPD (high polyamines) were transfected with mouse azin1 uCC-luciferase reporters. OF, last 10 codons of the uCC out-of-frame. Percent AUU initiation was calculated relative to corresponding AUG-initiated constructs. Error bars denote standard deviation; *p<0.05 (Student’s two-tailed t-test; n=4, assayed in duplicate). Results from tests of additional SPD concentrations are presented in Figure S1; relative reporter mRNA levels are presented in Table S1. (B) Schematic of the AZIN1 mRNA from mammals: conventional AUG initiated uORFs in cyan, uCC in red, and mORF in green. WebLogo representations of the most conserved C-terminal uCC residues from (top) 101 vertebrate AZIN1 orthologs or (middle) 320 metazoan ODC homologs, including the 101 vertebrate AZIN1 sequences; (bottom) WebLogo of the entire uCC from 34 Zygomycota ODC mRNAs. The highly conserved PPW motif is boxed. (C), azin1 uCC-Luc fusions with the indicated frameshift or point mutations (red) were tested as in panel (A). Mutations that alter the PPW motif are boxed. Firefly reporter values were normalized to a co-transfected Renilla luciferase initiated by AUG in perfect context. Error bars denote standard deviation; **p<0.01 (Student’s two-tailed t-test; n=5, assayed in duplicate). Results in panels (A) and (C) are from independent experiments; relative reporter mRNA levels are presented in Table S1.

Figure 2. Polyamine repression of AZIN1 mORF translation correlates with high ribosome occupancy near the 3′ end of the uCC

Ribosome protected mRNA fragments from HEK293T cells grown under low (DFMO, upper panel) and high (DFMO + 2 mM SPD, lower panel) polyamine conditions were mapped on the AZIN1 mRNA with the uCC (red) and mORF (green) depicted as rectangles in the schematic. The total (not normalized) ribosome fragment counts were aligned to the AZIN1 mRNA assuming a 15 nt offset from the 5′ end of the protected fragments to the A site. Quantified fragment counts mapping to the uCC and mORF under each condition are indicated above and below the ribosome profiles. (Inset) enlarged view of the fragments mapping within the last 15 codons of the uCC including on the PPW motif (orange).

Figure 3. Translation elongation pause enhances initiation at upstream AUU codon

(A) Amino acid sequence of the mouse azin1 uCC. The last 10 residues (red) were replaced by (bottom) the C-terminal two-thirds of the N. crassa AAP sequence (green) in uCC-AAP hybrid constructs; critical APP residue D12 is boxed in orange. (B) The indicated uCC-Luc reporters or a dual-luciferase human OAZ1 frameshift reporter (to monitor polyamine levels) were transfected either in HEK293T cells (right panel) pretreated with DFMO or DFMO + 3 mM SPD or in U2OS cells (middle panel) cultured in DMEM (400 μM Arg) media +/- 25 mM Arg. Percent AUU initiation calculation and firefly normalization were performed as in Figure 1. Error bars denote standard deviation; *p<0.05 (Student’s two-tailed t-test; n=4, assayed in duplicate); relative reporter mRNA levels are presented in Table S2.

Figure 4. High polyamine levels block eIF5A stimulation of PPW peptide synthesis

(A) Fractions of MEPPWK synthesis in yeast in vitro reconstituted elongation assays performed in the presence of the indicated concentration of eIF5A were quantified and fit to a single exponential equation. (B) Maximum fraction of peptide synthesis (Y_max) values from panel (A) were fit to the Michaelis-Menten equation to calculate the k_1/2 value for eIF5A (~0.1 μM). (C-F) Fractions of MEPPWK (C,E) or MEFFWK (D,F) synthesis in elongation assays performed in the presence of the indicated concentration of SPD, and either 0.1 μM (C,D) or 100 μM (E,F) eIF5A were plotted and fit to a single exponential equation or a simple two-step process for a sigmoidal curve (Lorsch and Herschlag, 1999). Biochemical data in panels (A-F) are representative of results obtained from three independent experiments. (G) HEK293T cells pretreated in 1 mM DFMO for 4 days were co-transfected with the indicated uCC-Luc reporters and either control or DHPS-targeted shRNA. After 48 hr, cells were supplemented with 1 μM SPD and then harvested after 24 hr. Percent AUU initiation calculation was performed as in Figure 1. Error bars denote standard deviation; ***p<0.001 (Student’s two-tailed t-test; n=4, assayed in duplicate).

Figure 5. Schematic model of polyamine-induced and eIF5A-mediated ribosomal queuing leading to enhanced initiation at the AUU start codon of the uCC on the AZIN1 mRNA

(A) Under conditions of lower polyamines most scanning 40S ribosomes skip over the uCC (red) start codon (AUU) without initiating and then initiate downstream on the AZIN1 mORF (green). The occasional ribosome that initiates on the uCC AUU start codon synthesizes the uCC peptide and then disengages from the mRNA. (B) Under conditions of higher polyamines any ribosome that initiates on the uCC start codon elongates down the uORF and encounters the PPW sequence. The high polyamines interfere with eIF5A function and cause the ribosome to stall. Subsequent scanning ribosomes, which mostly skip over the AUU start codon, and the occasional elongating ribosome form a queue behind the stalled ribosome. A queued subunit spends an extended time traversing and in the vicinity of the near cognate start codon allowing greater opportunities for initiation. The enhanced rate of initiation on the uCC reinforces the elongation stall, prevents ribosomes from scanning to the AZIN1 mORF, and effectively suppresses AZIN1 synthesis.

Figure 6. Ribosome queuing potentiates translation of the uCC to regulate AZIN1 mRNA translation

(A–C) Impairing 40S ribosome loading mitigates the effects of high polyamines on AZIN1 regulation. HEK293T cells were transfected with the indicated uCC-Luc, azin1 leader-Renilla or dual-luciferase OAZ1 reporter and incubated for 24 hr in DMEM supplemented with 1 mM aminoguanidine and 1 mM SPD in the absence or presence of 4EGI-1. Percent AUU initiation (A) was calculated as described in Figure 1A; relative reporter mRNA levels are presented in Table S3. To monitor AZIN1 synthesis (B), the full 5′ leader of mouse azin1 mRNA was fused upstream of Renilla luciferase. Wild type reporter activity was calculated as a percent of a reporter containing the out-of-frame uCC mutation and normalized to a co-transfected firefly luciferase reporter as in Figure 1C; relative reporter mRNA levels are presented in Table S3. To monitor levels of free polyamines, percent OAZ1 +1 frameshifting (C) was calculated relative to an in-frame reporter. Error bars denote standard deviation; **p<0.01 (Student’s two-tailed t-test; n=5, assayed in duplicate); # symbol in reporter names indicates that the AUG start codon of conventional uORFs were mutated to AAA. (D) Extending the length of the AZIN1 uCC impairs polyamine regulation. HEK293T cells were transfected with the indicated uCC-Luc reporters and grown as described in Figure 1A, except that cells were supplemented with 6 mM SPD. The grey box in the bottom two constructs indicates a 186 nt insertion (Table S6). In the middle construct the 62-codon extension starts with a non-initiating AAA codon and the uCC retains its normal AUU start codon; in the bottom construct the extension starts with an AUU codon and uCC start codon is changed to AAA. Luciferase activity of each reporter is compared to a reporter with AAA codons at both positions and the last 10 codons of the uCC out-of-frame. Error bars denote standard deviation; ***p<0.001 (Student’s two-tailed t-test; n=10, assayed in duplicate); relative reporter mRNA levels are presented in Table S3.