Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene.

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.

The Repertoire of RNA Modifications Orchestrates a Plethora of Cellular Responses.

Although a plethora of DNA modifications have been extensively investigated in the last decade, recent breakthroughs in molecular biology, including high throughput sequencing techniques, have enabled the identification of post-transcriptional marks that decorate RNAs; hence, epitranscriptomics has arisen. This recent scientific field aims to decode the regulatory layer of the transcriptome and set the ground for the detection of modifications in ribose nucleotides. Until now, more than 170 RNA modifications have been reported in diverse types of RNA that contribute to various biological processes, such as RNA biogenesis, stability, and transcriptional and translational accuracy. However, dysfunctions in the RNA-modifying enzymes that regulate their dynamic level can lead to human diseases and cancer. The present review aims to highlight the epitranscriptomic landscape in human RNAs and match the catalytic proteins with the deposition or deletion of a specific mark. In the current review, the most abundant RNA modifications, such as N6-methyladenosine (m6A), N5-methylcytosine (m5C), pseudouridine (Ψ) and inosine (I), are thoroughly described, their functional and regulatory roles are discussed and their contributions to cellular homeostasis are stated. Ultimately, the involvement of the RNA modifications and their writers, erasers, and readers in human diseases and cancer is also discussed.

The Transition from Cancer "omics" to "epi-omics" through Next- and Third-Generation Sequencing.

Deciphering cancer etiopathogenesis has proven to be an especially challenging task since the mechanisms that drive tumor development and progression are far from simple. An astonishing amount of research has revealed a wide spectrum of defects, including genomic abnormalities, epigenomic alterations, disturbance of gene transcription, as well as post-translational protein modifications, which cooperatively promote carcinogenesis. These findings suggest that the adoption of a multidimensional approach can provide a much more precise and comprehensive picture of the tumor landscape, hence serving as a powerful tool in cancer research and precision oncology. The introduction of next- and third-generation sequencing technologies paved the way for the decoding of genetic information and the elucidation of cancer-related cellular compounds and mechanisms. In the present review, we discuss the current and emerging applications of both generations of sequencing technologies, also referred to as massive parallel sequencing (MPS), in the fields of cancer genomics, transcriptomics and proteomics, as well as in the progressing realms of epi-omics. Finally, we provide a brief insight into the expanding scope of sequencing applications in personalized cancer medicine and pharmacogenomics.

Decoding the concealed transcriptional signature of the apoptosis-related BCL2 antagonist/killer 1 (BAK1) gene in human malignancies.

BCL2 antagonist/killer (BAK) is a multidomain pro-apoptotic effector protein, encoded by the human BAK1 gene, which has emerged as a key checkpoint in the apoptotic machinery. Disassembly of BAK's tertiary structure, such as the truncation of the α1 helix, leads to deregulation of the pro-apoptotic functions and reduction of the protein's stability, thus being implicated in human malignancies. Although many studies have already clarified the vital role of BAK in cellular mechanisms, its pre-mRNA maturation process under cancerous and physiological human cells is neglected. In the present work, we developed and employed a custom multiplexed nanopore sequencing approach that enabled the identification and structural characterization of previously undescribed BAK1 mRNA transcripts (BAK1 v.2-v.11). The described novel mRNAs are derived from multiple types of alternative splicing events, including exon skipping and intron retentions. The implemented multiplexed long-read sequencing approach provided the detailed expression profile of the novel mRNAs in a wide panel of human malignancies and at the same time allowed their relative quantification as compared to the annotated BAK1 v.1. The validation of each novel transcript was carried out with qPCR-based assays. Our results strongly support that most of the novel BAK1 mRNAs harbor open reading frames with conserved BH domains that provide new insights into the correlated mechanisms of apoptosis suppression and cancer. The current study highlights for the first time the hidden aspects of BAK1's transcriptional landscape in both physiological and cancerous human cells and distinguishes the amino acid sequence of the putative BAK isoforms that may possess key apoptosis-related functions not only in diseases, but also under normal cellular conditions.