An oncopeptide regulates m6A recognition by the m6A reader IGF2BP1 and tumorigenesis

Abstract

N⁶-methyladenosine (m⁶A) is the most prevalent modification in eukaryotic RNAs. The biological importance of m⁶A relies on m⁶A readers, which control mRNA fate and function. However, it remains unexplored whether additional regulatory subunits of m⁶A readers are involved in the m⁶A recognition on RNAs. Here we discover that the long noncoding RNA (lncRNA) LINC00266-1 encodes a 71-amino acid peptide. The peptide mainly interacts with the RNA-binding proteins, including the m⁶A reader IGF2BP1, and is thus named "RNA-binding regulatory peptide" (RBRP). RBRP binds to IGF2BP1 and strengthens m⁶A recognition by IGF2BP1 on RNAs, such as c-Myc mRNA, to increase the mRNA stability and expression of c-Myc, thereby promoting tumorigenesis. Cancer patients with RBRP^high have a poor prognosis. Thus, the oncopeptide RBRP encoded by LINC00266-1 is a regulatory subunit of m⁶A readers and strengthens m⁶A recognition on the target RNAs by the m⁶A reader to exert its oncogenic functions.

Fig. 1. The lncRNA LINC00266-1 encodes a 71-aa peptide, RBRP.

a The ORF-GFPmut constructs of ten indicated lncRNAs were transfected into HeLa cells and GFP fluorescence was detected. LOC* is LOC100126784 and FIRRE is also named LOC286467. Scale bar: 100 μm. b, c The indicated constructs were transfected into HeLa cells for 48 h, immunostaining of the RBRP-Flag fusion peptide was detected with anti-Flag and anti-RBRP antibodies (b), and levels of the RBRP-Flag fusion peptide were detected by performing western blottings with anti-Flag and anti-RBRP antibodies (c). Scale bar: 10 μm. d Two unique peptides in the RBRP peptide were identified using mass spectrometry. Source data are provided as a Source Data file.

Fig. 2. The LINC00266-1-encoded RBRP peptide is endogenously and naturally produced, and its upregulation is associated with a poor prognosis of patients with CRC.

a The RBRP peptide was immunostained with anti-RBRP antibodies in SW480 and SW620 cells. Scale bar: 10 μm. b The RBRP peptide levels were determined in the indicated cancer cell lines with different metastatic abilities (L: low; H: high). c Levels of the RBRP peptide were analyzed in five pairs of matched fresh primary CRC tissues (T) and their corresponding adjacent nontumoral colorectal tissues (NT). d Representative images of IHC staining for RBRP in CRC tissues (T) and their corresponding adjacent NT. e Differences in the RBRP scores between CRC tissues (T) and the corresponding adjacent NT are presented as a box plot with box & whiskers: 5–95 percentile (n = 90 tissue samples). Mann–Whitney U-test. f Relations between the RBRP levels and the percentage of patient death were analyzed in CRC samples (n = 39 CRC tissue samples with low RBRP and 51 samples with high RBRP). Pearson’s χ²-test. g A Kaplan–Meier analysis of the survival of patients with CRC according to RBRP scores. Log-rank test. RBRP scores of 0–3 were low, whereas scores of 4–7 were high. Source data are provided as a Source Data file.

Fig. 3. The RBRP peptide, but not the lncRNA LINC00266-1 itself, promotes tumorigenesis and metastasis in vitro and in vivo.

a–d The indicated LINC00266-1 (266) ORF-Flag (ORF), 5′UTR-ORF-Flag (5′U), and 5′UTR-ORFmut-Flag (MT or mut) constructs, which are resistant to anti-LINC00266-1 shRNA, were transfected into SW480 and HCT-116 cells with stable knockdown of LINC00266-1 expression by an anti-LINC00266-1 shRNA lentivirus (sh266); the RBRP level (a), cell growth (b) (n = 3 independent experiments), colony formation (c) (n = 3 independent experiments), and migration and invasion (d) (n = 5 independent experiments) were determined. Scale bar: 50 μm. e The in vivo tumorigenesis of the indicated cell lines stably expressing the indicated LINC00266-1 constructs was examined. Images and weights of the xenograft tumors are provided in the left and right panels, respectively (n = 6 mice per group). f NOD-SCID mice were injected with Luc-labeled HCT-116 cells (2 × 10⁶ cells/mouse) stably expressing the indicated LINC00266-1 constructs via the tail vein; luciferase activities were visualized at 9 weeks posttransplantation (n = 5 mice per group). g The fluorescence levels of mice in f were analyzed. Two-tailed unpaired Student’s t-test unless specifically stated, two-way ANOVA in b. The data are represented as the means ± SD. *p < 0.05, **p < 0.01, or ***p < 0.001, ns indicates no significance. Source data are provided as a Source Data file.

Fig. 4. RBRP interacts with the m⁶A reader IGF2BP1.

a Proteins that interacted with RBRP were identified by co-IP together with mass spectrometry. b, c The LINC00266-1 ORF-Flag (upper panel) and IGF2BP1-HA (lower panel) vectors were transfected into HEK293T cells, RBRP-Flag and IGF2BP1-HA complexes were co-IPed with anti-Flag and anti-HA antibodies in the absence (b) or presence (c) of RNase A treatment, and IGF2BP1 and RBRP/HA were detected, respectively. d Diagram of the different domains of the WT and mutated-IGF2BP1 constructs. e, f The indicated IGF2BP1-HA mutants were cotransfected with the LINC00266-1 ORF-Flag vector into HEK293T cells; RBRP-Flag (e) and IGF2BP1-HA (f) complexes were co-IPed with anti-Flag and anti-HA antibodies, respectively. The IGF2BP1-HA mutant and RBRP-Flag complexes were detected using anti-HA and anti-Flag antibodies, respectively. g, h The WT and GxxGΔ-mutated IGF2BP1-HA vectors were transfected into HEK293T cells and the interactions of RBRP with the IGF2BP1 GxxGΔ mutant were determined. i, j The WT or mutated RBRP-Flag constructs were cotransfected with the IGF2BP1-HA plasmid into HEK293T cells, and the interactions of the RBRP mutants with IGF2BP1 were determined. Source data are provided as a Source Data file.

Fig. 5. The RBRP oncopeptide, not the lncRNA LINC00266-1 itself, strengthens the recognition and binding of the m⁶A reader IGF2BP1 to m⁶A-modified c-Myc CRD mRNA.

a The in vitro binding of IGF2BP1 to m⁶A-unmethylated or -methylated c-Myc CRD mRNA oligos was investigated in HCT-116 cells stably expressing the LINC00266-1 ORF or 5′UTR-ORFmut (MT) by RNA pull-down assays. b The in vivo binding of IGF2BP1 on c-Myc CRD mRNA was determined in cells as in a by an RIP-qPCR assay (n = 3 independent experiments). c The m⁶A levels in IGF2BP1-bound RNAs were detected in cells as in a by dot blotting using an anti-m⁶A antibody. d WT or G19A-mutated RBRP-Flag vectors were transfected into HCT-116 cells with stable knockdown of LINC00266-1 expression (sh266); the in vitro binding of IGF2BP1 to m⁶A-unmethylated or -methylated c-Myc CRD mRNA oligos was analyzed by RNA pull-down assays. e The in vivo binding of IGF2BP1 on c-Myc CRD mRNA was determined in cells treated as in d by an RIP-qPCR assay (n = 3 independent experiments). f Recombinant IGF2BP1 protein, WT, or G19A-mutated RBRP peptide and m⁶A-methylated c-Myc CRD mRNA oligos were incubated, and the binding capability of IGF2BP1 on m⁶A-methylated c-Myc CRD mRNA was analyzed by an RNA EMSA assay. g The m⁶A levels in IGF2BP1-bound RNAs were detected in cells treated as in d by dot blotting. h The m⁶A writer METTL14 expression was silenced in HCT-116 cells stably expressing the LINC00266-1 ORF or 5′UTR-ORFmut (MT); the in vivo binding of IGF2BP1 on c-Myc CRD mRNA was determined by an RIP-qPCR assay (n = 3 independent experiments). i The WT or CRD-mutated c-Myc plasmids (sc-Myc), which were also synonymously mutated and resistant to anti-c-Myc siRNA, were cotransfected together with anti-c-Myc siRNAs into HCT-116 cells stably expressing the LINC00266-1 ORF; the in vivo binding of IGF2BP1 to c-Myc CRD mRNA was determined by an RIP-qPCR assay (n = 3 independent experiments). Two-tailed unpaired Student’s t-test. The data are represented as the means ± SD. *p < 0.05, **p < 0.01, or ***p < 0.001, ns indicates no significance. Source data are provided as a Source Data file.

Fig. 6. RBRP increases the stability and expression of c-Myc mRNA by regulating the m⁶A recognition by IGF2BP1 on c-Myc CRD mRNA.

a–c RBRP overexpression, but not the lncRNA LINC00266-1 itself, increases the half-life (a) (n = an experiment) and level (b) (n = 3 independent experiments) of c-Myc mRNA and the c-Myc protein level (c) in the cells stated in Fig. 5a. d, e The enhancement of the c-Myc mRNA half-life induced by RBRP (ORF) was blocked by silencing either the m⁶A reader IGF2BP1 (d) or the m⁶A writer METTL14 (e) in the cells stated in Fig. 5a (n = an experiment). f–h Cells were treated as in Fig. 5h. RBRP overexpression did not increase the c-Myc mRNA half-life (f) (n = an experiment) or level (h) (n = 3 independent experiments), or the c-Myc protein level (g) when A was mutated to U within six m⁶A consensus sites in c-Myc mRNA CRD. i, j The c-Myc protein (i) and mRNA (j) (n = 3 independent experiments) levels were determined in the CRC tissue samples shown in Fig. 2c. The c-Myc protein and mRNA levels were increased in CRC tissues (T) compared with those in the corresponding NT. k The c-Myc protein levels were positively correlated with the RBRP oncopeptide levels in the clinical tissue samples (n = 10 samples) using linear regression analysis. Two-tailed unpaired Student’s t-test unless specifically stated. The data are represented as the means ± SD. *p < 0.05, **p < 0.01, or ***p < 0.001, ns indicates no significance. Source data are provided as a Source Data file.

Fig. 7. RBRP regulates c-Myc mRNA stability and tumorigenesis by binding to IGF2BP1.

a–e The WT or G19A-mutated RBRP constructs, which were resistant to anti-LINC00266-1 shRNA, were transfected into HCT-116 cells with stable knockdown of LINC00266-1 expression by an anti-LINC00266-1 shRNA lentivirus targeting the 3′-UTR of the LINC00266-1 ORF (sh266); the half-life (a) (n = an experiment) and level (b) (n = 3 independent experiments) of c-Myc mRNA were detected, along with cell proliferation (c) (n = 3 independent experiments), colony formation (d) (n = 3 independent experiments), and migration and invasion (e) (n = 5 independent experiments). Scale bar: 50 μm. The G19A mutation of RBRP, which did not bind to the m⁶A reader IGF2BP1, abolished the stimulatory effects of RBRP on c-Myc stability and expression and cancer cell proliferation, colony formation, migration, and invasion. Two-tailed unpaired Student’s t-test unless specifically stated, two-way ANOVA in c. The data are represented as the means ± SD. *p < 0.05, **p < 0.01, or ***p < 0.001, ns indicates no significance. Source data are provided as a Source Data file.