G-quadruplex located in the 5′UTR of the BAG-1 mRNA affects both its cap-dependent and cap-independent translation through global secondary structure maintenance

Abstract The anti-apoptotic BAG-1 protein isoforms are known to be overexpressed in colorectal tumors and are considered to be potential therapeutic targets. The isoforms are derived from alternative translation initiations occuring at four in-frame start codons of a single mRNA transcript. Its 5′UTR also contains an internal ribosome entry site (IRES) regulating the cap-independent translation of the transcript. An RNA G-quadruplex (rG4) is located at the 5′end of the BAG-1 5′UTR, upstream of the known cis-regulatory elements. Herein, we observed that the expression of BAG-1 isoforms is post-transcriptionally regulated in colorectal cancer cells and tumors, and that stabilisation of the rG4 by small molecules ligands reduces the expression of endogenous BAG-1 isoforms. We demonstrated a critical role for the rG4 in the control of both cap-dependent and independent translation of the BAG-1 mRNA in colorectal cancer cells. Additionally, we found an upstream ORF that also represses BAG-1 mRNA translation. The structural probing of the complete 5′UTR showed that the rG4 acts as a steric block which controls the initiation of translation at each start codon of the transcript and also maintains the global 5′UTR secondary structure required for IRES-dependent translation.


BAG-1 endogenous RNA levels in CRC cell lines
The cDNAs resulting from the reverse-transcription (RT) of the total RNA extracted from diverse normal and cancerous colorectal cell lines were obtained from the J. Carrier biobank. The qPCR reactions were performed by the RNomics Platform as described in the main manuscript.

Western blot of endogenous BAG-1 in CRC cell lysates
Pooled protein lysates (10 µg) of the colorectal cell lines (HIEC, HCT116, CACO-2/15, SW48, DLD-1, HT-29, Colo205 and SNU) cultured under serum starvation condition were loaded on a 10 % SDS-PAGE gel which was migrated for 2 h 15 min at 150 V and transferred for 1 h at 100 V on a polyvinylidene difluoride (PVDF) membrane which was then blocked 15 min at room temperature in phoshate buffered saline (PBS) with 4% (w/v) nonfat drymilk (PBS-milk 4 %). The Western blot used to detect the BAG-1 protein isoforms was performed as described in the main manuscript, with the exception that β-actin was used as the loading control. After stripping of the membrane in 0.5 N NaOH twice for 10 min, and a thorough washing in PBS, the membrane was blocked in PBS-milk 4 % for 15 min and then incubated for 1 h at room temperature with the primary antibody, mouse anti-β-actin (AS441, Sigma), diluted 1: 1000 in the blocking buffer. After washes in PBS-T, the membrane was incubated for 1 h at room temperature with the secondary antibody, anti-mouse IgG (H+L) (IRDye 800CW, Li-Cor), diluted 1:10 000 in PBS-Milk 4 %. After 3 washes with PBS-T, the membrane was revealed using the Li-Cor Odyssey system.

Supplementary Figures and Legends
Supplementary Figure S1. BAG-1 protein isoforms' expression levels in the supplementary paired tissues samples of colorectal tumors at different stages and their adjacent healthy tissue (margin).
Protein expression levels, as measured by Western blot, of the three BAG-1 isoforms in the same pairs of margin (M)-tumor tissues (T) as in (Figure 2A). ERK2 is used as the loading control. The  Table   presenting the ratios of the G4mut constructs expression levels over their respective G4-WT construct from the luciferase assays, the RT-qPCR assays and the Western blots. The ratios are the means from the results obtained in the luciferase assays and RT-qPCR assays presented here and from the main manuscript ( Figure 4A-B, 5A-B, and Suppl. Fig 3A). The protein ratios are from the WB presented here only. Rluc expression levels were normalised over FLuc expression levels for the 3 types of assays. Increased expression from the G4mut constructions observed in the luciferase assays and WB does not arise only from transcriptional differences because both normalised luciferase expression and protein isoforms ratios are 1.3-fold higher than the RNA level ratio which suggest that the rG4 also affects post-transcriptional regulation, possibly translation.

Supplementary Tables
Supplementary Table S1. Clinicopathological parameters of the CRC patients.