Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

Rate and extent of pH decline affect proteolysis of cytoskeletal proteins and water-holding capacity in pork.

The objective of this study was to determine the extent to which early postmortem (PM) pH decline influences proteolysis of the intermediate filament protein desmin, the costameric proteins vinculin and talin and autolysis of µ-calpain in the longissimus muscle (LM) of pigs from two genetic lines. Based on the LM 3h pH (H=3h pH of LM>6.0; L=3h pH of LM pH<5.7) PM, 10 carcasses per line and pH group were selected. The average 3h pH within pH group was 6.23 (H) and 5.44 (L). The LM samples were collected 24, 48, 72, and 120h PM and percent drip loss was measured after 1, 2, and 4d of storage. Samples collected at 24, 48, 72, and 120h PM were used to monitor desmin, vinculin, and talin degradation and samples collected at 24h PM were used to determine the extent of µ-calpain autolysis by immunoblotting. Higher (P<0.01) pH values at 45min, 6h, and 24h PM and lower (P<0.01) drip losses after 1, 2, and 4d of storage were recorded in the H-compared to the L-group. Abundance of the 76kDa µ-calpain autolysis product was greater (P<0.01), proteolysis of talin at all measured time points and proteolysis of desmin after 24 and 48h PM was greater (P⩽0.03) in the H-group than in the L-group. The current findings indicate activation rate of µ-calpain may be associated with proteolysis of desmin and talin and could play a role in the development of drip loss. The rate of early PM pH decline can partly explain the variation of desmin and talin degradation by affecting the activation of µ-calpain.