Cellular stress alters the composition of the TRIB3 mRNA

potentiating protein production (Ref. I)

HepG2 human hepatocellular carcinoma cells were selected to be the subject of the TRIB3 mRNA isoform quantification, since these cells display readily detectable basal TRIB3 mRNA expression and robust TRIB3 induction in response to stress (Örd and Örd, 2005), and they have been used extensively as a model system for eIF2α–ATF4 pathway-based stress responses (for example, Shan et al., 2010). Additionally, the liver is a major site of TRIB3 expression in the body (discussed in section 2.3.1), and HepG2 cells have retained a considerable degree of hepatocyte metabolism and gene expression (Javitt, 1990; Tyakht et al., 2014). To activate ATF4-mediated cellular stress responses, the cells were treated with arsenite, a known inducer of oxidative stress, protein misfolding and eIF2α phosphorylation (Bernstam and Nriagu, 2000; Brostrom

et al., 1996; Zhou et al., 2008a).

The results reveal that in HepG2 cells incubated in normal growth medium, the predominant TRIB3 mRNA splice variant is 1A, which constitutes slightly over 90% of the TRIB3 mRNA population, and the remainder is mostly made up of 1B4 (8%), while the splice variants 1B1, 1B2 and 1B3 together account for less than 1% of the mRNA population. In response to arsenite treatment, the overall level of TRIB3 mRNA is upregulated 23-fold. The splice variants 1A, 1B1, 1B2 and 1B3 are all induced by around 20-fold, while splice variant 1B4 is upregulated more than 50-fold. Thus, in arsenite-treated cells, splice variant 1A remains predominant and 1B1–1B3 remain marginal, but the prevalence of 1B4 increases substantially (to 15%) in stressed cells as a consequence of the more prominent induction of 1B4 compared to other splice variants.

The TRIB3 C/EBP–ATF composite site, which mediates TRIB3 gene induc- tion in response to stress, is located in 33-bp tandem repeats that are situated 192 bp upstream of the exon 1A splice donor site. RT-qPCR results obtained with amplicons corresponding to different regions of exon 1A uncovered that under normal growth conditions, around half of the 1A mRNAs are generated using transcription start sites located upstream of the 33-bp repeat region, while

only around 10% are initiated downstream of the 33-bp repeats. These proportions are drastically altered in response to arsenite stress: the percentage of 1A mRNAs extending upstream of the 33-bp repeats is diminished (to approximately 3%) and the percentage of 1A mRNAs initiated downstream of the 33-bp repeats rises to above 80%. Thus, the upregulation of TRIB3 mRNA level in response to stress is mainly achieved by the production of splice variant 1A mRNAs with shortened 5′-UTRs, due to the preferential use of transcription start sites located downstream of the 33-bp repeat regions, while splice variant 1A mRNAs with long 5′-UTRs initiated upstream of the 33-bp repeats contribute significantly to the TRIB3 mRNA pool under basal conditions.

Results obtained by D. Örd (presented in Ref. I) reveal that splice variant 1A mRNAs with short 5′-UTRs are translated two-fold more efficiently in HepG2 cells than those with long 5′-UTRs. Strong mRNA secondary structures formed from GC-rich sequences in 5′-UTRs impair translation (Babendure et al., 2006; Kozak, 1989; Pickering and Willis, 2005), and both long and short forms of human TRIB3 splice variant 1A mRNA 5′-UTRs display nearly 80% GC con- tent, which is above the human average of around 60% (Kalari et al., 2006; Pesole et al., 2001). Additionally, the long forms of TRIB3 splice variant 1A 5′-UTRs contain two short ORFs that are situated upstream of the TRIB3 protein-coding ORF. These 5′-UTR elements, termed ‘upstream ORFs’ (uORFs), are present in more than 40% of human mRNAs (Peri and Pandey, 2001) and may inhibit the translation of the protein-coding ORF by directing ribosomes to initiate translation from the uORF start codon, provided that the uORF start codon is in a favorable nucleotide context for translation initiation (Kozak, 2001; Wang and Rothnagel, 2004). The initiation codon of one of the

TRIB3 uORFs is in an optimal context for translation initiation (Kozak, 1986;

Kozak, 1987), and further experiments performed by D. Örd (Ref. I) uncovered that the inclusion of this uORF underlies the different translational efficiencies of the long and short forms of TRIB3 splice variant 1A mRNAs.

Taken together, the results of Ref. I indicate that transcriptional and transla- tional mechanisms act in concert to facilitate the upregulation of TRIB3 in response to arsenite-induced cellular stress in HepG2 cells. In arsenite-treated cells, TRIB3 transcription initiation sites are shifted downstream compared to untreated cells, leading to an increased proportion of TRIB3 mRNAs with splice variant 1B4 and short 1A 5′-UTRs. As a result of this shift, stress-induced

TRIB3 transcription is predominantly initiated downstream of the 33-bp tandem

repeats which contain the stress-responsive C/EBP–ATF composite sites (described in section 2.3.3.1).

Variation of the first exon is often associated with alternative promoter usage (for example, Holthuizen et al., 1993; Hughes and Brady, 2005; Martineau et

al., 2004; Sobczak and Krzyzosiak, 2002), and the distribution of the TRIB3

transcription start sites over a range of roughly 500 bp is compatible with the concept of multiple core promoters, since human core promoters typically utilize transcription initiation sites spread over 50–100 bp (Sandelin et al., 2007). As discussed in section 2.3.3.1, C/EBP–ATF composite sites themselves

are flexible regarding positional requirements and satisfy the criteria for classification as enhancers. Arsenite-treated HepG2 cells preferentially induce

TRIB3 splice variant 1A mRNAs that contain a truncated 5′-UTR, which boosts

protein synthesis due to the lack of an inhibitory uORF. Elimination of an inhibitory uORF by 5′-UTR truncation has also been described for some other genes (Blaschke et al., 2003; Phelps et al., 1998).

Future studies quantifying TRIB3 mRNA isoforms are warranted in order to uncover the cell- and stress-type specificity of the TRIB3 mRNA variant profile. In addition to differentially containing uORFs, TRIB3 5′-UTRs are GC-rich and presumably impede translation also by forming secondary structures. It may be worth investigating whether TRIB3 mRNAs are subject to context-dependent derepression by increased translation initiation factor eIF4E activity, which alleviates the inhibitory effects of GC-rich 5′-UTRs (Koromilas et al., 1992; Nikolcheva et al., 2002; Rosenwald, 2004).

4.3. Trib3 expression in mouse bone marrow-derived