Like single-stranded RNA viruses, SARS-CoV-2 hijacks the host transcriptional machinery for its own replication. Numerous traditional differential gene expression-based investigations have examined the diverse clinical symptoms caused by SARS-CoV-2 infection. The virus, on the other hand, also affects the host splicing machinery, causing host transcriptional dysregulation, which can lead to diverse clinical outcomes. Hence, in this study, we performed host transcriptome sequencing of 125 hospital-admitted COVID-19 patients to understand the transcriptomic differences between the severity sub-phenotypes of mild, moderate, severe, and mortality. We performed transcript-level differential expression analysis, investigated differential isoform usage, looked at the splicing patterns within the differentially expressed transcripts (DET), and elucidated the possible genome regulatory features. Our DTE analysis showed evidence of diminished transcript length and diversity as well as altered promoter site usage in the differentially expressed protein-coding transcripts in the COVID-19 mortality patients. We also investigated the potential mechanisms driving the alternate splicing and discovered a compelling differential enrichment of repeats in the promoter region and a specific enrichment of SINE (Alu) near the splicing sites of differentially expressed transcripts. These findings suggested a repeat-mediated plausible regulation of alternative splicing as a potential modulator of COVID-19 disease severity. In this work, we emphasize the role of scarcely elucidated functional role of alternative splicing in influencing COVID-19 disease severity sub-phenotypes, clinical outcomes, and its putative mechanism. IMPORTANCE The wide range of clinical symptoms reported during the COVID-19 pandemic inherently highlights the numerous factors that influence the progression and prognosis of SARS-CoV-2 infection. While several studies have investigated the host response and discovered immunological dysregulation during severe infection, most of them have the common theme of focusing only up to the gene level. Viruses, especially RNA viruses, are renowned for hijacking the host splicing machinery for their own proliferation, which inadvertently puts pressure on the host transcriptome, exposing another side of the host response to the pathogen challenge. Therefore, in this study, we examine host response at the transcript-level to discover a transcriptional difference that culminates in differential gene-level expression. Importantly, this study highlights diminished transcript diversity and possible regulation of transcription by differentially abundant repeat elements near the promoter region and splicing sites in COVID-19 mortality patients, which together with differentially expressed isoforms hold the potential to elaborate disease severity and outcome.

There is a need to understand the molecular basis of testes under Non-Obstructive Azoospermia (NOA), a state of failed spermatogenesis. There has been a lack of attention to the transcriptome at the level of alternatively spliced mRNAs (iso-mRNAs) and the mechanism of gene expression regulation. Hence, we aimed to establish a reliable iso-mRNA profile of NOA-testes, and explore molecular mechanisms - especially those related to gene expression regulation. We sequenced mRNAs from testicular samples of donors with complete spermatogenesis (control samples) and a failure of spermatogenesis (NOA samples). We identified differentially expressed genes and their iso-mRNAs via standard NGS data analyses. We then listed these iso-mRNAs hierarchically based on the extent of consistency of differential quantities across samples and groups, and validated the lists via RT-qPCRs (for 80 iso-mRNAs). In addition, we performed extensive bioinformatic analysis of the splicing features, domains, interactions, and functions of differentially expressed genes and iso-mRNAs. Many top-ranking down-regulated genes and iso-mRNAs, i.e., those down-regulated more consistently across the NOA samples, are associated with mitosis, replication, meiosis, cilium, RNA regulation, and post-translational modifications such as ubiquitination and phosphorylation. Most down-regulated iso-mRNAs correspond to full-length proteins that include all expected domains. The predominance of alternative promoters and termination sites in these iso-mRNAs indicate their gene expression regulation via promoters and UTRs. We compiled a new, comprehensive list of human transcription factors (TFs) and used it to identify TF-'TF gene' interactions with potential significance in down-regulating genes under the NOA condition. The results indicate that RAD51 suppression by HSF4 prevents SP1-activation, and SP1, in turn, could regulate multiple TF genes. This potential regulatory axis and other TF interactions identified in this study could explain the down-regulation of multiple genes in NOA-testes. Such molecular interactions may also have key regulatory roles during normal human spermatogenesis.

The delayed diagnosis of pancreatic cancer has resulted in rising mortality rate and low survival rate that can be circumvented using potent theranostics biomarkers. The treatment gets complicated with delayed detection resulting in lowered 5-year relative survival rate. In our present study, we employed systems biology approach to identify central genes that play crucial roles in tumor progression. Pancreatic cancer genes collected from various databases were used to construct a statistically significant interactome with 812 genes that was further analysed thoroughly using topological parameters and functional enrichment analysis. The significant genes in the network were then identified based on the maximum degree parameter. The overall survival analysis indicated through hazard ratio [HR] and gene expression [log Fold Change] across pancreatic adenocarcinoma revealed the critical role of FN1 [HR 1.4; log2(FC) 5.748], FGA [HR 0.78; log2(FC) 1.639] FGG [HR 0.9; log2(FC) 1.597], C3 [HR 1.1; log2(FC) 2.637], and QSOX1 [HR 1.4; log2(FC) 2.371]. The functional significance of the identified hub genes signified the enrichment of integrin cell surface interactions and proteoglycan syndecan-mediated cell signaling. The differential expression, low overall survival and functional significance of FN1 gene implied its possible role in controlling metastasis in pancreatic cancer. Furthermore, alternate splice variants of FN1 gene showed 10 protein coding transcripts with conserved cell attachment site and functional domains indicating the variants' potential role in pancreatic cancer. The strong association of the identified hub-genes can be better directed to design potential theranostics biomarkers for metastasized pancreatic tumor.

Alternative ribonucleic acid (RNA) splicing can lead to the assembly of different protein isoforms with distinctive functions. The outcome of alternative splicing (AS) can result in a complete loss of function or the acquisition of new functions. There is a gap in knowledge of abnormal RNA splice variants promoting cancer stem cells (CSCs), and their prospective contribution in cancer progression. AS directly regulates the self-renewal features of stem cells (SCs) and stem-like cancer cells. Notably, octamer-binding transcription factor 4A spliced variant of octamer-binding transcription factor 4 contributes to maintaining stemness properties in both SCs and CSCs. The epithelial to mesenchymal transition pathway regulates the AS events in CSCs to maintain stemness. The alternative spliced variants of CSCs markers, including cluster of differentiation 44, aldehyde dehydrogenase, and doublecortin-like kinase, α6β1 integrin, have pivotal roles in increasing self-renewal properties and maintaining the pluripotency of CSCs. Various splicing analysis tools are considered in this study. LeafCutter software can be considered as the best tool for differential splicing analysis and identification of the type of splicing events. Additionally, LeafCutter can be used for efficient mapping splicing quantitative trait loci. Altogether, the accumulating evidence re-enforces the fact that gene and protein expression need to be investigated in parallel with alternative splice variants.

Small open reading frames (small ORFs/sORFs/smORFs) are potentially coding sequences smaller than 100 codons that have historically been considered junk DNA by gene prediction software and in annotation screening; however, the advent of next-generation sequencing has contributed to the deeper investigation of junk DNA regions and their transcription products, resulting in the emergence of smORFs as a new focus of interest in systems biology. Several smORF peptides were recently reported in noncanonical mRNAs as new players in numerous biological contexts; however, their relevance is still overlooked in coding potential analysis. Hence, this review proposes a smORF classification based on transcriptional features, discussing the most promising approaches to investigate smORFs based on their different characteristics. First, smORFs were divided into nonexpressed (intergenic) and expressed (genic) smORFs. Second, genic smORFs were classified as smORFs located in noncoding RNAs (ncRNAs) or canonical mRNAs. Finally, smORFs in ncRNAs were further subdivided into sequences located in small or long RNAs, whereas smORFs located in canonical mRNAs were subdivided into several specific classes depending on their localization along the gene. We hope that this review provides new insights into large-scale annotations and reinforces the role of smORFs as essential components of a hidden coding DNA world.

A considerable number of deposited variants has provided new possibilities for knowledge discovery in different types of prostate cancer. Here, we analyzed variants located on 3'UTR, 5'UTR, CDs, Intergenic, and Intronic regions in castration-resistant prostate cancer (8496 variants), familial prostate cancer (3241 variants), metastatic castration-resistant prostate cancer (3693 variants), and prostate cancer (16599 variants). Chromosome regions 10p15-p14 and 2p13 were highly enriched (P < 0.00001) for variants located in 3'UTR, 5'UTR, CDs, intergenic, and intronic regions in castration-resistant prostate cancer. In contrast, 10p15-p14, 10q23.3, 12q13.11, 13q12.3, 1q25, and 8p22 regions were enriched (P < 0.001) in familial prostate cancer. In metastatic castration-resistant prostate cancer, 10p15-p14, 10q23.3, 11q22-q23, 14q21.1, and 14q32.13 were highly variant regions (P < 0.001). Chromosome 2 and chromosome 1 hosted many enriched variant regions. AKR1C3, BRCA1, BRCA2, CHGA, CYP19A1, HOXB13, KLK3, and PTEN contained the highest number of 3'UTR, 5'UTR, CDs, Intergenic, and Intronic variants. Network analysis showed that these genes are upstream of important functions including prostate gland development, tumor recurrence, prostate cancer-specific survival, tumor progression, cancer mortality, long-term survival, cancer recurrence, angiogenesis, and AR. Interestingly, all of EGFR, JAK2, NR3C1, PDZD2, and SEMA3C genes had single nucleotide polymorphisms (SNP) in castration-resistant prostate cancer, consistent with high selection pressure on these genes during drug treatment and consequent resistance. High occurrence of variants in 3'UTRs suggests the importance of regulatory variants in different types of prostate cancer; an area that has been neglected compared with coding variants. This study provides a comprehensive overview of genomic regions contributing to different types of prostate cancer.