Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members

The complicity of human RAS proteins in cancer is a well-documented fact, both due to the mutational hyperactivation of these GTPases and the overexpression of the genes encoding these proteins. Thus, it can be easily assumed that the study of RAS genes at the transcriptional and post-transcriptional level is of the utmost importance. Although previous research has shed some light on the basic mechanisms by which GTPases are involved in tumorigenesis, limited information is known regarding the transcriptional profile of the genes encoding these proteins. The present study highlights for the first time the wide spectrum of the mRNAs generated by the three most significant RAS genes (KRAS, NRAS and HRAS), providing an in-depth analysis of the splicing events and exon/intron boundaries. The implementation of a versatile, targeted nanopore-sequencing approach led to the identification of 39 novel RAS mRNA transcript variants and to the elucidation of their expression profiles in a broad panel of human cell lines. Although the present work unveiled multiple hidden aspects of the RAS gene family, further study is required to unravel the biological function of all the novel alternative transcript variants, as well as the putative protein isoforms.

AZFa Y Gene, DDX3Y, Evolved Novel Testis Transcript Variants in Primates with Proximal 3´UTR Polyadenylation for Germ Cell Specific Translation

Translational control is a major level of gene expression regulation in the male germ line. DDX3Y located in the AZFa region of the human Y chromosome encodes a conserved RNA helicase important for translational control at the G1-S phase of the cell cycle. In human, DDX3Y protein is expressed only in premeiotic male germ cells. In primates, DDX3Y evolved a second promoter producing novel testis-specific transcripts. Here, we show primate species-specific use of alternative polyadenylation (APA) sites for the testis-specific DDX3Y transcript variants. They have evolved first in the 3´UTRs of primate DDX3Y transcripts. A distal APA site is used for polyadenylation of DDX3Y testis transcripts in Callithrix jacchus; two proximal APAs in Macaca mulatta, in Pan trogloydates and in human. This shift corresponds with a significant increase of DDX3Y protein expression in the macaque testis and kidney tissue. In chimpanzee and human, shift to predominant use of the most proximal APA site is associated with translation of these DDX3Y transcripts in only premeiotic male germ cells. We therefore assume evolution of a positive selection process for functional DDX3Y testis transcripts in these primates to promote increase of their stability and balancing translation efficiency especially in the male germ line.

Neuron-Specific Mechanisms Control the Mitochondrial Regulator PGC-1a

Mitochondrial dysfunction has been proposed as a hallmark of the aging process. Specifically, as a function of aging, mitochondria appear to have decreased enzyme activity and respiratory capacity and increase reactive oxygen species production. Brain aging is associated with morphological and homeostatic changes, including alterations in brain size, cognitive impairment, and white and grey matter integrity. However, the causes of these changes remain an open and actively pursued field of study. The ubiquitously expressed transcriptional coactivator peroxisome proliferator-activated receptor gamma-coactivator 1 (PGC-1a) has been described as the master regulator of mitochondrial function. Despite the emerging connections between PGC-1a and disease vulnerability, the regulation of PGC-1a outside of the skeletal muscle, liver, and adipose tissue is not well defined. This is particularly true in the brain, where PGC-1a is enriched in neurons, and alterations in expression levels have been linked to neurodegenerative disorders. Here we report that astrocytes and neurons differ substantially in mitochondrial status and the transcript variants of PGC-1a expressed, including using a neuron-specific promoter. Taking advantage of the ability of the tau-kinase GSK-3b to influence PGC-1a expression, we investigate how transcript variants are differentially regulated in primary neurons and astrocytes. Neuronal PGC-1a responds robustly to GSK3b inhibition by lithium, switching the dominant promoter, leading to changes in isoform distribution and abundance, while astrocytes are refractory to lithium treatment. The data presented here highlight key mechanisms for neuron-specific metabolic regulation that are likely to be relevant to neurodegenerative diseases that have a link to mitochondrial dysfunction.

Pan-tissue and -cancer analysis of ROR1 and ROR2 transcript variants identify novel functional significance for an alternative splice variant of ROR1

Background: ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far. Methods: ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues. Results: ROR1/2 have four and eight transcript variants respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2. Conclusion: ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.

Characterization of aquaporin1b (AQP1b) mRNA in mud loach (Misgurnus mizolepis) in response to heavy metal and immunostimulant stimuli

Aquaporins (AQPs) facilitate the transport of water or other small solutes into cells in the presence of osmotic gradients. However, the current understanding of piscine AQP gene with cellular stress responses has been still limitedly exemplified. In present study, we characterized the mud loach AQP1b gene at the nucleotide and amino acid levels. We identified three AQP 1b transcript variants (mmAQP1b_tv1, mmAQP1b_tv2, and mmAQP1b_tv3). Then, we examined the AQP1b promoter region and observed several transcription factor binding sites (TFBS) for nuclear factor of activated T-cells (NFAT), SRY-box, c-AMP responsive element binding protein (CREB), GATA binding factor, and hepatic nuclear factor-1. Interestingly, mmAQP1b transcription was differentially modulated by heavy metal or immunostimulant challenge. Further studies to deepen the knowledge of fish AQP-mediated adaptation response potentially relevant to molecular pathogenesis are warranted.Summary statementWe identified mud loach AQP1b transcript variants and consensus sequences involved in stress or innate immunity in promotor region. AQP1b transcription was differentially modulated by heavy metal or immunostimulant challenge.

Computational Methods to Study Human Transcript Variants in COVID-19 Infected Lung Cancer Cells

Microbes and viruses are known to alter host transcriptomes by means of infection. In light of recent challenges posed by the COVID-19 pandemic, a deeper understanding of the disease at the transcriptome level is needed. However, research about transcriptome reprogramming by post-transcriptional regulation is very limited. In this study, computational methods developed by our lab were applied to RNA-seq data to detect transcript variants (i.e., alternative splicing (AS) and alternative polyadenylation (APA) events). The RNA-seq data were obtained from a publicly available source, and they consist of mock-treated and SARS-CoV-2 infected (COVID-19) lung alveolar (A549) cells. Data analysis results show that more AS events are found in SARS-CoV-2 infected cells than in mock-treated cells, whereas fewer APA events are detected in SARS-CoV-2 infected cells. A combination of conventional differential gene expression analysis and transcript variants analysis revealed that most of the genes with transcript variants are not differentially expressed. This indicates that no strong correlation exists between differential gene expression and the AS/APA events in the mock-treated or SARS-CoV-2 infected samples. These genes with transcript variants can be applied as another layer of molecular signatures for COVID-19 studies. In addition, the transcript variants are enriched in important biological pathways that were not detected in the studies that only focused on differential gene expression analysis. Therefore, the pathways may lead to new molecular mechanisms of SARS-CoV-2 pathogenesis.

Exploring Evidence of Non-coding RNA Translation With Trips-Viz and GWIPS-Viz Browsers

Detection of translation in so-called non-coding RNA provides an opportunity for identification of novel bioactive peptides and microproteins. The main methods used for these purposes are ribosome profiling and mass spectrometry. A number of publicly available datasets already exist for a substantial number of different cell types grown under various conditions, and public data mining is an attractive strategy for identification of translation in non-coding RNAs. Since the analysis of publicly available data requires intensive data processing, several data resources have been created recently for exploring processed publicly available data, such as OpenProt, GWIPS-viz, and Trips-Viz. In this work we provide a detailed demonstration of how to use the latter two tools for exploring experimental evidence for translation of RNAs hitherto classified as non-coding. For this purpose, we use a set of transcripts with substantially different patterns of ribosome footprint distributions. We discuss how certain features of these patterns can be used as evidence for or against genuine translation. During our analysis we concluded that the MTLN mRNA, previously misannotated as lncRNA LINC000116, likely encodes only a short proteoform expressed from shorter RNA transcript variants.

Differential Effects of WRAP53 Transcript Variants on the Biological Behaviours of Human Non-Small Cell Lung Cancer Cells

Background: The WD40-encoding RNA antisense to p53 (WRAP53) gene, an antisense gene of TP53, has 3 different transcriptional start sites that yield 3 transcript variants. One of these variants WRAP53-1β encodes a WD repeat-containing protein WRAP53β, whereas WRAP53-1α is a noncoding RNA that regulates p53 mRNA levels. These variants are involved in the progression of non-small cell lung cancer (NSCLC). However, how the different transcript variants regulate NSCLC cell behaviours is to be elucidated.Methods: Wild-type p53 NSCLC A549 cells and p53-mutated H1975 cells were transfected with WRAP53-1α and WRAP53-1β siRNAs, and their behaviours were examined colony formation, cell viability, apoptosis, cell cycle, wound healing, and cell invasion assays.Results: WRAP53-1α knockdown increased the mRNA and protein levels of p53, whereas depletion of WRAP53-1β had no effect on p53 expression. WRAP53-1α knockdown suppressed colony formation and proliferation of A549 cells, but had the opposite effects on H1975 cells. However, WRAP53-1β knockdown promoted A549 cell growth. Depletion of WRAP53-1α and WRAP53-1β promoted apoptosis in A549 but not H1975 cells. WRAP53-1α knockdown increased the proportion of A549 but not H1975 cells at the G0/G1 phase. However, WRAP53-1β knockdown decreased the proportion of cells at the G0/G1 phase in A549 cells. Depletion of WRAP53-1α suppressed A549 cell migration and invasion, and promoted H1975 cell migration and invasion. However, depletion of WRAP53-1β had the opposite effects.Conclusions: The 2 WRAP53 transcript variants exerted opposite functions in NSCLC cells and regulated NSCLC cell behaviours in a p53-dependent manner.