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1. Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

   https://doi.org/10.1186/s12885-022-10311-1  

   Lo, April ; McSharry, Maria ; Berger, Alice H. ( December 2022 , BMC Cancer)

   Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.

   We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.

   Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRAS^G12Vand KRAS^Q61Hcells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45^M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.

   Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

    

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2. CPGView : A package for visualizing detailed chloroplast genome structures

   https://doi.org/10.1111/1755-0998.13729  

   Liu, Shengyu ; Ni, Yang ; Li, Jingling ; Zhang, Xinyi ; Yang, Heyu ; Chen, Haimei ; Liu, Chang ( January 2023 , Molecular Ecology Resources)

   Chloroplast genomes have been widely used in studying plant phylogeny and evolution. Several chloroplast genome visualization tools have been developed to display the distribution of genes on the genome. However, these tools do not draw features, such as exons, introns, repetitive elements, and variable sites, disallowing in‐depth examination of the genome structures. Here, we developed and validated a software package called Chloroplast Genome Viewers (CPGView). CPGView can draw three maps showing (i) the distributions of genes, variable sites, and repetitive sequences, including microsatellites, tandem and dispersed repeats; (ii) the structure of the cis‐splicing genes after adjusting the exon‐intron boundary positions using a coordinate scaling algorithm, and (iii) the structure of the trans‐splicing generps12. To test the accuracy of CPGView, we sequenced, assembled, and annotated 31 chloroplast genomes from 31 genera of 22 families. CPGView drew maps correctly for all the 31 chloroplast genomes. Lastly, we used CPGView to examine 5998 publicly released chloroplast genomes from 2513 genera of 553 families. CPGView succeeded in plotting maps for 5882 but failed to plot maps for 116 chloroplast genomes. Further examination showed that the annotations of these 116 genomes had various errors needing manual correction. The test on newly generated data and publicly available data demonstrated the ability of CPGView to identify errors in the annotations of chloroplast genomes. CPGView will become a widely used tool to study the detailed structure of chloroplast genomes. The web version of CPGView can be accessed fromhttp://www.1kmpg.cn/cpgview.

    

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3. The Arabidopsis INNER NO OUTER ( INO ) gene acts exclusively and quantitatively in regulation of ovule outer integument development

   https://doi.org/10.1002/pld3.485  

   Skinner, Debra J. ; Dang, Trang ; Gasser, Charles S. ( February 2023 , Plant Direct)

   TheINNER NO OUTER(INO) gene is essential for formation of the outer integument of ovules inArabidopsis thaliana. Initially described lesions inINOwere missense mutations resulting in aberrant mRNA splicing. To determine the null mutant phenotype, we generated frameshift mutations and found, in confirmation of results on another recently identified frameshift mutation, that such mutants have a phenotype identical to the most severe splicing mutant (ino‐1), with effects specific to outer integument development. We show that the altered protein of aninomRNA splicing mutant with a less severe phenotype (ino‐4) does not haveINOactivity, and the mutant is partial because it produces a small amount of correctly splicedINOmRNA. Screening for suppressors ofino‐4in a fast neutron‐mutagenized population identified a translocated duplication of theino‐4gene, leading to an increase in the amount of this mRNA. The increased expression led to a decrease in the severity of the mutant effects, indicating that the amount ofINOactivity quantitatively regulates outer integument growth. The results further confirm that the role ofINOin Arabidopsis development is specific to the outer integument of ovules where it quantitatively affects the growth of this structure.

    

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4. 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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5. Comprehensive analysis of TCGA data reveals correlation between DNA methylation and alternative splicing

   https://doi.org/10.1186/s12864-022-08992-w  

   Lin, Shuting ; Yi, Soojin ; Qiu, Peng ( November 2022 , BMC Genomics)

   The effect of DNA methylation on the regulation of gene expression has been extensively discussed in the literature. However, the potential association between DNA methylation and alternative splicing is not understood well. In this study, we integrated multiple omics data types from The Cancer Genome Atlas (TCGA) and systematically examined the relationship between DNA methylation and alternative splicing. Using the methylation data and exon expression data, we identified many CpG sites significantly associated with exon expression in various types of cancers. We further observed that the direction and strength of significant CpG-exon correlation tended to be consistent across different cancer contexts, indicating that some CpG-exon correlation patterns reflect fundamental biological mechanisms that transcend tissue- and cancer- types. We also discovered that CpG sites correlated with exon expressions were more likely to be associated with patient survival outcomes compared to CpG sites that did not correlate with exon expressions. Furthermore, we found that CpG sites were more strongly correlated with exon expression than expression of isoforms harboring the corresponding exons. This observation suggests that a major effect of CpG methylation on alternative splicing may be related to the inclusion or exclusion of exons, which subsequently impacts the relative usage of various isoforms. Overall, our study revealed correlation patterns between DNA methylation and alternative splicing, which provides new insights into the role of methylation in the transcriptional process.

    

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