METTL3 regulates breast cancer-associated alternative splicing switches.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer. N6-methyladenosine (m6A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro. We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m6A-methyltransferase in tumorigenesis. Specifically, we find that both m6A deposition in splice site boundaries and in splicing and transcription factor transcripts, such as MYC, direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

m6A RNA Immunoprecipitation Followed by High-Throughput Sequencing to Map N6-Methyladenosine.

N6-methyladenosine (m6A) is the most abundant internal modification on messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) in eukaryotes. It influences gene expression by regulating RNA processing, nuclear export, mRNA decay, and translation. Hence, m6A controls fundamental cellular processes, and dysregulated deposition of m6A has been acknowledged to play a role in a broad range of human diseases, including cancer. m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq or m6A-seq) is a powerful technique to map m6A in a transcriptome-wide level. After immunoprecipitation of fragmented polyadenylated (poly(A)+) rich RNA by using specific anti-m6A antibodies, both the immunoprecipitated RNA fragments together with the input control are subjected to massively parallel sequencing. The generation of such comprehensive methylation profiles of signal enrichment relative to input control is necessary in order to better comprehend the pathogenesis behind aberrant m6A deposition.

5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.