ALYREF recruits ELAVL1 to promote colorectal tumorigenesis via facilitating RNA m5C recognition and nuclear export

Abstract

ALYREF can recognize 5-methylcytosine (m5C) decoration throughout RNAs to regulate RNA metabolism. However, its implications in cancer and precise regulatory mechanisms remain largely elusive. Here, we demonstrated that ALYREF supported colorectal cancer (CRC) growth and migration. Integrated analysis of ALYREF-RIP-Bis-seq and transcriptome profiles identified ribosomal protein S6 kinase B2 (RPS6KB2) and regulatory-associated protein of mTOR (RPTOR) as ALYREF's possible downstream effectors. Mechanistically, ALYREF formed a complex with ELAV like RNA binding protein 1 (ELAVL1) to cooperatively promote m5C recognition and nuclear export of the two mRNAs. Moreover, ALYREF protein was highly expressed in tumor tissues of CRC patients, which predicted their poor prognosis. E2F transcription factor 6 (E2F6)-mediated transactivation gave a molecular insight into ALYREF overexpression. Collectively, ALYREF recruits ELAVL1 to collaboratively facilitate m5C recognition and nuclear export of RPS6KB2 and RPTOR transcripts for colorectal tumorigenesis, providing RNA m5C methylation as promising therapeutic targets and prognostic biomarkers for CRC.

Fig. 1. ALYREF is required for the growth and migration capacity of CRC.

a–d CRC cells were exposed to specific siRNAs or plasmids. RNA level, protein level, cell viability and migration capability were detected by RT-qPCR (a), immunoblot (b), CCK-8 (c) and Transwell (d) assays, respectively. e HCT116 cells transduced with lentivirus vectors were subcutaneously inoculated into Balb/c nude mice (n = 5). Tumors were stripped, weighed and measured three weeks after the implantations. Data were presented as the mean ± SD. ALY ALYREF. **P < 0.01; ***P < 0.001; two-sided Student’s t test.

Fig. 2. RPS6KB2 and RPTOR are mTOR-related downstream transcripts of ALYREF.

a Transcriptome analysis was conducted in DLD-1 cells treated with siALYREF or siNC. KEGG pathway enrichment analysis revealed the top five significant ALYREF-activated pathways and oncogenes annotated in the mTOR pathway. b Venn plot showing the overlay of ALYREF-RIP-bis-seq data from Yang et al., ALYREF-upregulated genes and oncogenes annotated in mTOR signaling from our RNA-seq data. c, d Transcripts screened were reverified with RT-qPCR and immunoblot analysis. RPS6KB2 and RPTOR encode S6K2 and Raptor protein, respectively. e, f Cell proliferation and migration capabilities in rescue assays were assessed by CCK8 (e) and Transwell (f) techniques, respectively. Data were presented as the mean ± SD. ALY ALYREF. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant; two-tailed Student’s t test.

Fig. 3. ALYREF serves as an m5C reader to export RPS6KB2 and RPTOR transcripts.

a ALYREF-RIP-Bis-seq from Yang et al. screened ALYREF-recognized m5C sites in the target transcripts. b–d Lysates from naïve or transfected cells were subjected to RIP assays with antibodies against ALYREF or control IgG. The RIP enrichment of a certain sample was normalized to its input. e Lysates from siRNA-transfected cells were subjected to m5C-RIP assays using anti-m5C or control anti-IgG antibodies. Transcripts of interest were detected by specific primers covering m5C sites. The m5C enrichment of each sample was expressed as the fold change in the m5C-RIP group normalized to the IgG-RIP group. f, g Biotin-labeled RNA probes against full-length CDS, their antisense sequences and fragments were subjected to RNA pull-down assays. The protein of interest was detected by anti-ALYREF antibody. h pmirGLO reporter plasmids (RPS6KB2-wt/mut or RPTOR-wt/mut) were co-transfected with plasmids as indicated for dual luciferase reporter assays. The relative luciferase activity was calculated as the ratio of firefly luciferase signal to Renilla luciferase signal and normalized to siNC. i After siRNA silencing, the nucleocytoplasmic distribution of RPS6KB2 and RPTOR mRNAs was measured by RT-qPCR following subcellular fractionation. Data were presented as the mean ± SD. ALY ALYREF; wt wild type; mut mutant. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant; two-sided Student’s t test.

Fig. 4. The ELAVL1 protein directly interacts with the ALYREF protein and target mRNAs.

a ALYREF-ELAVL1 protein interaction was predicted by STRING online tool. b, c Exogenous co-IP assays were conducted with anti-Flag M2 (b) or anti-HA (c) agarose beads. d, e Endogenous co-IP assays were conducted with protein A/G magnetic beads to capture primary antibodies against ALYREF (d) or ELAVL1 (e). f, g GST pull-down assays were performed with purified His-tagged protein and spin columns containing GST-tagged proteins or GST alone. h Lysates from naïve cells were subjected to RIP assays with antibodies against ELAVL1 or control IgG. The RIP enrichment of a certain sample was normalized to its input. i, j Biotin-labeled RNA probes against full-length CDS, their antisense sequences and fragments were subjected to RNA pull-down assays. The protein of interest was detected by an anti-ELAVL1 antibody.

Fig. 5. ALYREF recruits ELAVL1 to cooperatively facilitate m5C recognition and export target transcripts for colorectal tumorigenesis.

a control and treated CRC cells were harvested for RIP assays with anti-ALYREF or control anti-IgG antibodies. RIP enrichment of certain samples was quantified and normalized to the input. b, c pmirGLO reporter plasmids (RPS6KB2-wt/mut or RPTOR-wt/mut) were co-transfected with plasmids/siRNAs as indicated for dual luciferase reporter assays. The relative luciferase activity of each sample was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity and normalized to siNC. d–g RNA nucleocytoplasmic shuttling, RNA expression level, growth ability and migration capability were quantified by subcellular fractionation (d), RT-qPCR (e), CCK-8 (f) and Transwell (g) techniques in rescue experiments, respectively. Data were presented as the mean ± SD. ALY ALYREF. Rescue, siALYREF + ELAVL1. **P < 0.01; ***P < 0.001; two-sided Student’s t test.

Fig. 6. E2F6 serves as an upstream transcriptional activator of ALYREF.

a, b Candidate transcriptional factors bound to the ALYREF promoter were predicted in silico (a) and verified with RT-qPCR (b). c Schematic plot of the putative E2F6 binding site in the ALYREF promoter. d Naïve cells were harvested for ChIP assays with anti-E2F6 or anti-IgG antibodies. ChIP enrichment was quantified by RT-qPCR with specific primers for the ALYREF promoter and normalized to the input. e pGL3 reporter plasmid carrying the ALYREF-wt/mut promoter was co-transfected with Renilla reporter plasmid and siRNAs as indicated for dual luciferase reporter assays. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity and normalized to siNC. f, g Growth and migration capabilities were quantified by CCK-8 (f) and Transwell (g) techniques in rescue experiments, respectively. Data were presented as the mean ± SD. ALY ALYREF. ***P < 0.001; ns not significant; two-sided Student’s t test.

Fig. 7. ALYREF is highly expressed in CRC patients with poor outcomes.

a–f ALYREF and ELAVL1 protein expression in CRC tissues and matched normal tissues from a tissue microarray was assessed using IHC. Typical images (a, d) are shown, together with expression (b, e), survival (c) and correlation results (f). Scale bar, 50 µm. Paired t test in (b, e), Log-rank test in (c) and Pearson’s correlation test in (f). g Schematic presentation of the ALYREF/ELAVL1/m5C axis in RNA export and colorectal tumorigenesis. E2F6-transactivated ALYREF interacted with ELAVL1 to cooperatively facilitate m5C recognition and nuclear export of RPS6KB2 and RPTOR transcripts, thereby increasing their expressions and tumor growth in CRC.