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1. PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation

   https://doi.org/10.1093/toxsci/kfaa158  

   Aluru, Neelakanteswar ; Karchner, Sibel I ( October 2020 , Toxicological Sciences)

   null (Ed.)

   Abstract Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3′ UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate 5%), respectively. The majority of the peaks were preferentially located around the 3′ UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels. 

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2. APA-Scan: detection and visualization of 3′-UTR alternative polyadenylation with RNA-seq and 3′-end-seq data

   https://doi.org/10.1186/s12859-022-04939-w  

   Fahmi, Naima Ahmed ; Ahmed, Khandakar Tanvir ; Chang, Jae-Woong ; Nassereddeen, Heba ; Fan, Deliang ; Yong, Jeongsik ; Zhang, Wei ( September 2022 , BMC Bioinformatics)

   The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3′-untranslated region (3′-UTR) of mRNA produces transcripts with shorter or longer 3′-UTR. Often, 3′-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3′-UTR APA is known to modulate translation and provides a mean to regulate gene expression at the post-transcriptional level. Current bioinformatics pipelines have limited capability in profiling 3′-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3′-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3′-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations.

   APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3′-UTR transcripts in the RNA-seq data. APA-Scan works in three major steps: (i) calculate the read coverage of the 3′-UTR regions of genes; (ii) identify the potential APA sites and evaluate the significance of the events among two biological conditions; (iii) graphical representation of user specific event with 3′-UTR annotation and read coverage on the 3′-UTR regions. APA-Scan is implemented in Python3. Source code and a comprehensive user’s manual are freely available athttps://github.com/compbiolabucf/APA-Scan.

   APA-Scan was applied to both simulated and real RNA-seq datasets and compared with two widely used baselines DaPars and APAtrap. In simulation APA-Scan significantly improved the accuracy of 3′-UTR APA identification compared to the other baselines. The performance of APA-Scan was also validated by 3′-end-seq data and qPCR on mouse embryonic fibroblast cells. The experiments confirm that APA-Scan can detect unannotated 3′-UTR APA events and improve genome annotation.

   APA-Scan is a comprehensive computational pipeline to detect transcriptome-wide 3′-UTR APA events. The pipeline integrates both RNA-seq and 3′-end-seq data information and can efficiently identify the significant events with a high-resolution short reads coverage plots.

    

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3. Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing

   https://doi.org/10.1038/s41467-023-41207-8  

   Zhang, Zhiping ; Bae, Bongmin ; Cuddleston, Winston H. ; Miura, Pedro ( September 2023 , Nature Communications)

   Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. Here, the coordination of cassette exon (CE) splicing and APA inDrosophilawas investigated using a targeted long-read sequencing approach we call Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3’ ends. Using PL-Seq, we identified genes that exhibit significant differences in CE splicing depending on connectivity to short versus long 3’UTRs. Genomic long 3’UTR deletion was found to alter upstream CE splicing in short 3’UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3’UTRs. This work highlights the importance of considering connectivity to alternative 3’UTRs when monitoring AS events.

    

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4. Structured 3′ UTRs destabilize mRNAs in plants

   https://doi.org/10.1186/s13059-024-03186-x  

   Zhang, Tianru ; Li, Changhao ; Zhu, Jiaying ; Li, Yanjun ; Wang, Zhiye ; Tong, Chun-Yip ; Xi, Yu ; Han, Yi ; Koiwa, Hisashi ; Peng, Xu ; et al ( February 2024 , Genome Biology)

   RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3′ untranslated regions (3′ UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3′ UTRs in gene expression in different organisms and/or contexts.

   Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3′ UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3′ UTR and, resultantly, a poorly structured 3′ UTR. RNA decay assays indicate that poorly structured 3′ UTRs could promote mRNA stability, whereas highly structured 3′ UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3′ UTRs’ RSS and transcript accumulation in the transcriptomes ofArabidopsis, rice, and even human. Mechanistically, transcripts with highly structured 3′ UTRs are preferentially degraded by 3′–5′ exoribonuclease SOV and 5′–3′ exoribonuclease XRN4, leading to decreased expression inArabidopsis. Finally, we engineer different structured 3′ UTRs to an endogenousFTgene and alter theFT-regulated flowering time inArabidopsis.

   We conclude that highly structured 3′ UTRs typically cause reduced accumulation of the harbored transcripts inArabidopsis. This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3′ UTRs’ RSS to modify plant traits in agricultural production and mRNA stability in biotechnology.

    

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5. APA-Scan: Detection and Visualization of 3’-UTR APA with RNA-seq and 3’-end-seq Data

   https://doi.org/10.1101/2020.02.16.951657  

   Fahmi, Naima Ahmed ; Chang, Jae-Woong ; Nassereddeen, Heba ; Ahmed, Khandakar Tanvir ; Fan, Deliang ; Yong, Jeongsik ; Zhang, Wei ( January 2020 , BioRxiv)

   The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3’-untranslated region (3’-UTR) of mRNA produces transcripts with shorter 3’-UTR. Often, 3’-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3’-UTR APA provides a means to regulate gene expression at the post-transcriptional level and is known to promote translation. Current bioinformatics pipelines have limited capability in profiling 3’-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3’-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3’-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations. APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3’-UTR transcripts in the RNA-seq data. The performance of APA-Scan was validated by qPCR. 

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