Background: N6-methyladenosine (m6A), a prevalent mRNA modification, is dynamically regulated by methyltransferases, including METTL3 and METTL14. Materials & methods: In the current study, we employed a custom hybrid-seq method to identify novel METTL3/14 transcripts, explore their protein-coding capacities and predict the putative role of the METTL isoforms. Results: Demultiplexing of the hybrid-seq barcoded datasets unraveled the expression patterns of the newly identified mRNAs in major malignancies as well as in non-malignant cells, providing a deeper understanding of the methylation pathways. Open reading frame query revealed novel METTL3/14 isoforms, broadening our perspective for the structural diversity within METTL family. Conclusion: Our findings offer significant insights into the intricate transcriptional landscape of METTL3/14, shedding light on the regulatory mechanisms underlying methylation in mRNAs. Nanopore sequencing unveils novel alternative METTL3 and METTL14 mRNAs. In total, eight novel METTL3 mRNAs (METTL3 v.2–v.9) and eight novel METTL14 mRNAs (METTL14 v.2–v.9) were identified. Expression profiling of the METTL3/14 mRNAs was performed in human malignant and non-cancerous cells. METTL3 v.9 exhibits significantly higher expression levels across the investigated human malignancies. METTL14 v.5 emerged as the most abundant alternative mRNA in the tested cancer types. METTL3 v.2–v.9 and METTL14 v.2–v.9 were successfully quantified through variant-specific qPCR assays. Ribosome profiling supports that specific METTL3/14 mRNAs are highly promising to encode protein isoforms. In silico structural analysis highlights the methyltransferase activity in most of the described putative novel isoforms.

Background: N6-methyladenosine (m6A), a prevalent mRNA modification, is dynamically regulated by methyltransferases, including METTL3 and METTL14. Materials & methods: In the current study, we employed a custom hybrid-seq method to identify novel METTL3/14 transcripts, explore their protein-coding capacities and predict the putative role of the METTL isoforms. Results: Demultiplexing of the hybrid-seq barcoded datasets unraveled the expression patterns of the newly identified mRNAs in major malignancies as well as in non-malignant cells, providing a deeper understanding of the methylation pathways. Open reading frame query revealed novel METTL3/14 isoforms, broadening our perspective for the structural diversity within METTL family. Conclusion: Our findings offer significant insights into the intricate transcriptional landscape of METTL3/14, shedding light on the regulatory mechanisms underlying methylation in mRNAs. Nanopore sequencing unveils novel alternative METTL3 and METTL14 mRNAs. In total, eight novel METTL3 mRNAs (METTL3 v.2–v.9) and eight novel METTL14 mRNAs (METTL14 v.2–v.9) were identified. Expression profiling of the METTL3/14 mRNAs was performed in human malignant and non-cancerous cells. METTL3 v.9 exhibits significantly higher expression levels across the investigated human malignancies. METTL14 v.5 emerged as the most abundant alternative mRNA in the tested cancer types. METTL3 v.2–v.9 and METTL14 v.2–v.9 were successfully quantified through variant-specific qPCR assays. Ribosome profiling supports that specific METTL3/14 mRNAs are highly promising to encode protein isoforms. In silico structural analysis highlights the methyltransferase activity in most of the described putative novel isoforms.

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 – sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 – sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.