Regulation of translational fidelity during UPR by regulating the expression of C/D box snoRNPs

Background: Endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed ER capacity, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Activation of PERK during ER stress phosphorylates eIF2α, which regulates protein synthesis by inhibiting global translation initiation and ribosome biogenesis through downregulating ribosomal RNA (rRNA) synthesis [1]. Small nucleolar ribonucleoproteins (snoRNPs) are small RNAs complexed with diverse sets of proteins, and play multiple roles in ribosomes biogenesis. There are two major types of snoRNPs: Box C/D and Box H/ACA. Box C/D snoRNPs catalyze the methylation of the 2′ hydroxyl group of ribose in RNA, while Box H/ACA snoRNPs catalyze the isomerization of uridine to pseudouridine in rRNA [2]. The methylations and pseudouridylations of rRNAs can fine tune protein synthesis.. Here we evaluated the expression snoRNPs during UPR and effect of UPR and knock-down of FBL on translation fidelity.

Methods: FBL-deficient subclones were produced via tet-on shRNA system, and were validated through PCR. Cell viability and proliferation were determined using MTS and colony formation, respectively. Translation fidelity (such as alterations in nonsense suppression, frameshifts, ribosome pausing and translation initiation by IRES) was determined using report assays.

Results: TNM plots showed that the expressions of NOP56, NOP58, NOP10, DKC1, GAR1, NHP2 and SNU13 were significantly upregulated in invasive breast cancer. KM plots revealed that breast cancer patients with higher levels of NOP56, NOP58, NOP10, DKC1 and NHP2 showed a trend towards poorer Relapse-free survival (RFS). Interestingly, NOP56 was remarkably correlated with worse RFS in Luminal A and B breast cancers, whereas it showed no significance in basal and HER2+ subtypes. In both MCF7 and T47D cell lines, NOP56 was significantly downregulated after TG or BFA treatment, and FBL and NHP2L1 were also remarkably downregulated after TG treatment. Western blot showed that BFA and TG suppressed NOP56 expression in MCF7 and T47D. Moreover, FBL regulates rRNA methylation including XBP1 pause 2A, IRES dependent translation inition, frame shifting and 190 UGA non-sense stop. Knock-down of FBL in MCF7 cells can suppress IRES-dependent translation initiation, and increase non-sense suppression, indicating that FBL plays an important role in ribosome function in breast cancer.

Conclusions: NOP56 can act as a oncogenic factor and play a role in UPR pathway in breast cancer. FBL may enchance IRES-dependent translation initiation through ribosome function in breast cancer.

Keywords: snoRNP; NOP56; FBL; breast cancer; Riosome methylation; UPR pathway.

Reference

1. DuRose, J.B., et al., Phosphorylation of eukaryotic translation initiation factor 2alpha coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress. Mol Cell Biol, 2009, 29(15), 4295-4307.

2. Massenet, S., Bertrand, E. and Verheggen, C., Assembly and trafficking of box C/D and H/ACA snoRNPs. Rna Biol, 2017, 14(6), 680-692.