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1. Dichotomous intronic polyadenylation profiles reveal multifaceted gene functions in the pan- cancer transcriptome

   Sun, Jiao; Kim, Jin-Young; Jun, Semo; Park, Meeyeon; Jong, Ebbing de; Chang, Jae-Woong; Cheng, Sze; Fan, Deliang; Chen, Yue; Griffin, Timothy J; et al (October 2024, Experimental & Molecular Medicine)

   Alternative cleavage and polyadenylation within introns (intronic APA) generate shorter mRNA isoforms; however, their physiological significance remains elusive. In this study, we developed a comprehensive workflow to analyze intronic APA profiles using the mammalian target of rapamycin (mTOR)-regulated transcriptome as a model system. Our investigation revealed two contrasting effects within the transcriptome in response to fluctuations in cellular mTOR activity: an increase in intronic APA for a subset of genes and a decrease for another subset of genes. The application of this workflow to RNA-seq data from The Cancer Genome Atlas demonstrated that this dichotomous intronic APA pattern is a consistent feature in transcriptomes across both normal tissues and various cancer types. Notably, our analyses of protein length changes resulting from intronic APA events revealed two distinct phenomena in proteome programming: a loss of functional domains due to significant changes in protein length or minimal alterations in C- terminal protein sequences within unstructured regions. Focusing on conserved intronic APA events across 10 different cancer types highlighted the prevalence of the latter cases in cancer transcriptomes, whereas the former cases were relatively enriched in normal tissue transcriptomes. These observations suggest potential, yet distinct, roles for intronic APA events during pathogenic processes and emphasize the abundance of protein isoforms with similar lengths in the cancer proteome. Furthermore, our investigation into the isoform-specific functions of JMJD6 intronic APA events supported the hypothesis that alterations in unstructured C-terminal protein regions lead to functional differences. Collectively, our findings underscore intronic APA events as a discrete molecular signature present in both normal tissues and cancer transcriptomes, highlighting the contribution of APA to the multifaceted functionality of the cancer proteome. 

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2. mTOR-regulated U2af1 tandem exon splicing specifies transcriptome features for translational control

   https://doi.org/10.1093/nar/gkz761  

   Chang, Jae-Woong; Yeh, Hsin-Sung; Park, Meeyeon; Erber, Luke; Sun, Jiao; Cheng, Sze; Bui, Alexander M; Fahmi, Naima Ahmed; Nasti, Ryan; Kuang, Rui; et al (September 2019, Nucleic Acids Research)

   Abstract U2 auxiliary factor 1 (U2AF1) functions in 3′-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms’ inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3′-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5′-untranslated region (5′-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5′-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control. 

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

   https://doi.org/10.3390/ijms22189684  

   Sun, Jiao; Fahmi, Naima Ahmed; Nassereddeen, Heba; Cheng, Sze; Martinez, Irene; Fan, Deliang; Yong, Jeongsik; Zhang, Wei (September 2021, International Journal of Molecular Sciences)

   null (Ed.)

   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. 

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4. Alternative splicing preferentially increases transcript diversity associated with stress responses in the extremophyte Schrenkiella parvula

   Wijesinghege, Chathura; Tran, Kieu-Nga; Dassanayake, Maheshi (October 2022, bioRxiv)

   Alternative splicing extends the coding potential of genomes by creating multiple isoforms from one gene. Isoforms can render transcript specificity and diversity to initiate multiple responses required during transcriptome adjustments in stressed environments. Although the prevalence of alternative splicing is widely recognized, how diverse isoforms facilitate stress adaptation in plants that thrive in extreme environments are unexplored. Here we examine how an extremophyte model, Schrenkiella parvula, coordinates alternative splicing in response to high salinity compared to a salt-stress sensitive model, Arabidopsis thaliana. We use Iso-Seq to generate full length reference transcripts and RNA-seq to quantify differential isoform usage in response to salinity changes. We find that single-copy orthologs where S. parvula has a higher number of isoforms than A. thaliana as well as S. parvula genes observed and predicted using machine learning to have multiple isoforms are enriched in stress associated functions. Genes that showed differential isoform usage were largely mutually exclusive from genes that were differentially expressed in response to salt. S. parvula transcriptomes maintained specificity in isoform usage assessed via a measure of expression disorderdness during transcriptome reprogramming under salt. Our study adds a novel resource and insight to study plant stress tolerance evolved in extreme environments. 

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5. Comparative Transcriptomics Provides Insights into Reticulate and Adaptive Evolution of a Butterfly Radiation

   https://doi.org/10.1093/gbe/evz202  

   Zhang, Wei; Leon-Ricardo, Brian X; van Schooten, Bas; Van Belleghem, Steven M; Counterman, Brian A; McMillan, W Owen; Kronforst, Marcus R; Papa, Riccardo; Chang, Belinda (October 2019, Genome Biology and Evolution)

   Abstract Butterfly eyes are complex organs that are composed of a diversity of proteins and they play a central role in visual signaling and ultimately, speciation, and adaptation. Here, we utilized the whole eye transcriptome to obtain a more holistic view of the evolution of the butterfly eye while accounting for speciation events that co-occur with ancient hybridization. We sequenced and assembled transcriptomes from adult female eyes of eight species representing all major clades of the Heliconius genus and an additional outgroup species, Dryas iulia. We identified 4,042 orthologous genes shared across all transcriptome data sets and constructed a transcriptome-wide phylogeny, which revealed topological discordance with the mitochondrial phylogenetic tree in the Heliconius pupal mating clade. We then estimated introgression among lineages using additional genome data and found evidence for ancient hybridization leading to the common ancestor of Heliconius hortense and Heliconius clysonymus. We estimated the Ka/Ks ratio for each orthologous cluster and performed further tests to demonstrate genes showing evidence of adaptive protein evolution. Furthermore, we characterized patterns of expression for a subset of these positively selected orthologs using qRT-PCR. Taken together, we identified candidate eye genes that show signatures of adaptive molecular evolution and provide evidence of their expression divergence between species, tissues, and sexes. Our results demonstrate: 1) greater evolutionary changes in younger Heliconius lineages, that is, more positively selected genes in the cydno–melpomene–hecale group as opposed to the sara–hortense–erato group, and 2) suggest an ancient hybridization leading to speciation among Heliconius pupal-mating species. 

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