GLIOBLASTOMAS ARE CHARACTERIZED BY A REDUCED NUMBER OF M6A SITES IN LNCRNAs

Recent studies have suggested the potential role of posttranscriptional N6-methyladenosine (m6A) RNA modifications in diverse biological processes, including tumor development. In contrast to intensively studied mRNA modifications, insufficient information is known so far about the epitranscriptome of long non-coding RNAs (lncRNAs). Furthermore, an epitranscriptome of LncRNAs in glioma pathogenesis has not been previously presented. Thus, this study aimed to profile m6A modifications within lncRNAs in different grades of gliomas using direct RNA long-read sequencing, with the goal of yielding both nucleotide and transcript-resolved m6A hits as biomarkers for tumor progression. Here we explored ONT direct RNA-seq to detect N6-methyladenosine (m6A) sites within lncRNAs' RRACH motifs in 26 glioma tumor tissues. Generally, 98.5% of m6A-modified RRACH motifs were detected in mRNA transcripts, and 1.16% in lncRNAs. We identified 60-748 unique m6A-modified lncRNAs for individual gliomas. On average, 15.84% of all RRACH motifs within lncRNAs were modified in glioblastomas (GB), while the m6A frequency reached 23.73% in the low-grade glioma (LGG) group. Unsupervised clustering analysis based on lncRNAs' m6A status resulted in three m6A clusters. Patients within the highly modified lncRNAs cluster (C3) experienced significantly longer survival compared to patients with lower methylation rates who were clustered into C1/C2 (2901 days in C3 vs 439 days in C1, log-rank test p=0.019).  In summary, we discovered that lncRNAs are highly modified in LGG while multiple epi-marks, found in low-grade gliomas, are absent in GB tissues revealing m6A contribution to glioma pathology.