RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation

Background: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells.

Methods: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot.

Results: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found.

Conclusion: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.