Role of DDX3X in translation regulation in acute ER stress
In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation Overall design: We prepared 10%-50% sucrose gradients in polyallomer tubes for SW-41 rotor using the Gradient Master 108, and collected the polysome profiles using the Piston Fractionator with a TRIAX Flowcell (260 nm) (BioComp). 293FT cells were seeded in 100-mm culture dishes (3 dishes per polysome profile sample) and grown to 80% confluency. 1.5 h prior to collection, cell culture media was replaced with full media containing DMSO (non-stressed condition) or 400 nM Thapsigargin (acute ER stress). Cells were incubated for the last 5 min of the treatment with cycloheximide (CHX) at a final concentration of 100 µg/mL. Cells were then washed twice with ice cold phosphate-buffered saline supplemented with CHX and centrifuged at 4,500 X g for 5 min at 4° C. Cell pellets were lysed in 800 µL of Hypotonic buffer (5 mM Tris HCl pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 0.5% Triton X, 0.5% sodium deoxycholate, 100 µg/mL CHX, 2 mM DTT, 200 units/mL RNase inhibitor, 1X protease inhibitor cocktail). Cells were resuspended in the hypotonic buffer either by pipetting or vortexing for 5 sec followed by centrifugation at 17,000 xg for 7 min at 4° C. The supernatant (post-nuclear lysate) OD was measured at 260 nm to determine the amount of lysate needed to be added to sucrose gradient. Samples of the post-nuclear lysates were also kept for western blots and input RNA extraction 500 mL from the top of the sucrose gradient was removed and the lysate volume per condition was adjusted so they contain the same OD between 10 to 20 at 260 nm and added to the top of the sucrose gradient. Each gradient was weighed and balanced before ultracentrifugation at 222,222 xg for 2 hours at 4° C while using low brake option. 14 fractions of 800 µL were collected. Immediately after polysome fractions were collected, 400 µL of each fraction was mixed with equal volume TRIZOL LS (Thermo) and kept at -80C. RNA extraction was carried out by manufacturer's instructions. RNA was treated with RQ1 DNase (Promega), followed by phenol extraction and ethanol precipitation. The concentration and the purity of RNA was measured using nanodrop and pure RNA with a 260/280 ratio within 1.8 - 2.0 was acquired. To prepare polyA mRNA RNA-Seq libraries, approximately 800 ng of high-quality total RNA was used per sample. The quality was determined with a Nanodrop spectrophotometer, and only RNA that had a OD260/280 of 1.8-2.0 was used. We followed the Illumina Stranded mRNA Prep, Ligation Reference Guide, Document # 1000000124518 V03 protocol without changes.