Hypoxia‐dependent mRNA expression pattern of splicing factor YT521 and its impact on oncological important target gene expression

Abstract

The ubiquitously expressed splicing factor YT521 (YTHDC1) is characterized by alternatively spliced isoforms with regulatory impact on cancer-associated gene expression. Our recent findings account for the prognostic significance of YT521 in endometrial cancer. In this study, we investigated the hypoxia-dependency of YT521 expression as well as its differential isoform activities on oncological important target genes. YT521's potential regulatory influence on splicing was investigated by a minigene assay for the specific target gene CD44. Functional splicing analysis was performed by YT521 knock-down or overexpression, respectively. In addition, YT521 expression was determined under hypoxia. The two protein-generating YT521 mRNA isoforms 1 and 2 caused a comparable, specific induction of CD44v alternative splicing (P < 0.01). In a number of oncological target genes, YT521 upregulation significantly altered BRCA2 expression pattern, while YT521 knock-down created a significant regulatory impact on PGR expression, respectively. Hypoxia induced a specific switch towards the processing of two non-protein-coding mRNA variants, of which one is described for the first time in this study. The presented study underlines the comparable regulatory potential of both YT521 isoforms 1 and 2, on the investigated target genes in vivo and in vitro. Hypoxia induces a specific switch in YT521 expression pattern towards the two non-protein coding mRNA variants, the already characterized isoform 3 and the newly discovered exon 8-skipping isoform. The altered YT521 alternative splicing is functionally coupled with nonsense-mediated decay and can be interpreted as regulated unproductive splicing and transcription with consecutive impact on the processing of specific cancer-associated genes, such as BRCA2 and PGR.

Constitutive patterns of gene expression regulated by RNA-binding proteins

“…We hypothesize that perturbation of the regulation by YTHDC1 of potentially oncogenic genes such as RET, PRMT2, RARG and HOXA9 could be involved in the pathogenesis of related tumors. In fact, experimental studies support the implications for YTHDC1 in cancer progression with regard to angiogenesis, growth factor signaling, immortalization, genetic instability, tissue invasion and apoptosis [59,64,65]. …”

The RNA Modification N 6-Methyladenosine and Its Implications in Human Disease

“…Exposure of breast cancer cells to hypoxia also leads to the upregulation of ZNF217, a transcription factor known to interact with METTL3 and inhibit its function [77] (Figure 2D). In gynecological tumor cell lines, hypoxia leads to a reduction of YTHDC1 protein levels, through changes in splicing that generate mRNA isoforms targeted by nonsense-mediated decay [78]. The m 6 A reader YTHDC2 [79] has been shown to promote translation of HIF-1 α and contributes to colon tumor metastasis [80].…”

YT521 promotes metastases of endometrial cancer by differential splicing of vascular endothelial growth factor A

“…Previous studies have demonstrated YT521 as an important RNA splicing-related protein, and CD44 is one of the identified targets for YT521. Nevertheless, the biological [18,19]. However, whether YT521 may similarly regulate the splicing of VEGF-A in EC is unknown.…”

“…Primary antibodies were anti-VEGF-A, anti-α-tubulin (R&D System, Los Angeles, CA, USA), and anti-YT521 [18,19]. α-Tubulin was used as a protein loading control for analyzing YT521 levels.…”

“…Nevertheless, the biological effects of YT521 in cancer are poorly understood. Recently, we have shown high YT521 levels in EC patients with a poor prognosis [18,19]. Moreover, YT521 seemed to regulate the RNA splicing of CD44 [20,21].…”