scholarly journals Epigenetic loss of heterozygosity of Apc and an inflammation-associated mutational signature detected in Lrig1+/--driven murine colonic adenomas

2019 ◽  
Author(s):  
Jessica Preston
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Loss Of Heterozygosity ◽  
Somatic Mutations ◽  
Rna Binding ◽  
Point Mutations ◽  
Colonic Adenomas ◽  
A Genome ◽  
Guanine Adducts ◽  
Splicing Patterns

AbstractMurine colonic adenomas induced by the loss of a single copy of the tumor suppressor gene Apc in Lrig1+/- expressing progenitor cells grow rapidly, with high penetrance and tumor multiplicity. This study investigates the prevalence of intertumoral genetic heterogeneity and phenotypic variation across tumors, and attempts to identify the genomic cause of the unusual phenotype. Adult Lrig1-CreERT2/+; Apc-flox/+ mice were intraperitoneal injected with 2mg tamoxifen for 3 consecutive days which induced highly penetrant, dysplastic adenomas in the distal colon ∼100 days later. Whole tumors (n=14) from 8 mice were excised and tumor exome DNA and mRNA were sequenced. Somatic mutations present in the tumor exome DNA were compared with adjacent normal colon (n=9 tumors from 3 mice). Putative somatic mutations were called after stringent filtering using SeuratSomatic, a Genome Analysis Toolkit software module. RNA-Seq was performed on tumor mRNA (n=5 tumors from 5 mice) compared to wildtype colon (n=3). Differential gene expression was profiled using the R package DESeq2. Copy number variations and splicing defects were assessed using custom tracks on the UCSC genome browser.Adenomas resulting from the loss of Apc in Lrig1+/-- expressing colonic progenitor cells are genetically heterogeneous and hypermutated, containing ∼25-30 high-quality somatic mutations per megabase. A loss of heterozygosity of Apc was not observed in the tumor genomes, however evidence of an epigenetic loss of heterozygosity was readily apparent in the tumor transcriptome. The tumors display a strong bias toward G: C > A: T point mutations, which are a signature of guanine adducts, associated with tobacco tar and H. pylori infections. Putative tumor-driving mutations were detected and thousands of differentially expressed genes were identified including several UDP glucuronosyltransferases. Abnormal splicing patterns characterized by a loss of intron retention were detected in several RNA-binding genes throughout the tumor transcriptome. The widespread defects in gene expression, genomic stability, and splicing patterns implies that an early epigenetic loss of Apc in Lrig1+/--expressing progenitor cells causes a rapid formation of guanine adducts and a corresponding accumulation of mutation C>A point mutations. This study demonstrates that randomly-appearing oncogenic mutations can become fixed into a latent genomic reservoir prior to advanced disease.

scholarly journals Visualizing the translation and packaging of HIV-1 full-length RNA

2020 ◽  
Vol 117 (11) ◽  
pp. 6145-6155 ◽  
Author(s):  
Jianbo Chen ◽  
Yang Liu ◽  
Bin Wu ◽  
Olga A. Nikolaitchik ◽  
Preeti R. Mohan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Rna Binding ◽  
Virus Assembly ◽  
Full Length ◽  
Rna Molecules ◽  
Rna Translation ◽  
Translation Template ◽  
A Genome ◽  
Hiv 1

HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation.

scholarly journals Splicing Control in Normal and Aberrant Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-47-SCI-47
Author(s):  
Esther Obeng
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Iron Transport ◽  
Rna Binding ◽  
Intron Retention ◽  
Point Mutations ◽  
Cytoskeletal Proteins ◽  
Mrna Splicing ◽  
Splice Sites ◽  
Erythroid Maturation

Abstract Alternative splicing is employed by all eukaryotic cells to increase proteome diversity and to regulate gene expression. RNA sequencing analysis of purified populations of erythroblasts at different stages of maturation has led to the identification of a dynamic alternative splicing program that directly modulates the protein isoform expression of cytoskeletal proteins and genes involved in RNA processing, heme biosynthesis, and iron transport. Regulated interactions of multiple RNA-binding proteins and cis-regulatory sequences located within exons or their flanking introns promote or inhibit functional spliceosome assembly at splice junctions, leading to altered exon inclusion or intron retention. Exon skipping regulates tissue and stage specific isoform expression of red cell membrane cytoskeletal proteins including EPB41, ankyrin, and band 3. Intron retention can lead to a frame shift during translation and introduction of a premature termination codon (PTC), that marks the transcript for degradation via the nonsense mediated decay pathway (NMD) upon export from the nucleus into the cytoplasm. Intron retention leading to posttranscriptional regulation of gene expression during terminal erythroid maturation has been identified in genes involved in RNA processing and iron transport including SF3B1, SNRNP70, SLC25A37 and SLC25A28. Mutations that alter mRNA splice sites or introduce PTCs lead to a variety of congenital anemias including beta thalassemia, hereditary pyropoikilocytosis, hereditary elliptocytosis, and hereditary spherocytosis. Aberrant mRNA splicing has subsequently been shown to lead to acquired anemias in subsets of patients with myelodysplastic syndromes (MDS). Somatic missense mutations in components of the spliceosome are the most common category of mutations in MDS. These point mutations lead to changes in the RNA binding specificity of the involved proteins and aberrant splicing of a subset of transcripts. Mutant SF3B1, the most commonly mutated splicing factor in MDS, has been shown to cause aberrant pre-mRNA splicing and an increase in transcripts predicted to undergo NMD due to use of upstream, cryptic 3' splice sites. Our group and others evaluating the strong genotype-phenotype association between SF3B1 point mutations and subtypes of MDS with ring sideroblasts have shown that the expression of the mitochondrial iron transporter, ABCB7, is decreased in samples from SF3B1-mutant MDS patients due to cryptic 3' splice site selection and introduction of a PTC between exons 8 and 9. The identification and functional validation of additional aberrantly spliced mutant-SF3B1 target genes is ongoing, with the goal of understanding how point mutations in a core component of the mRNA splicing machinery can lead to such specific effects on erythroid maturation. Disclosures No relevant conflicts of interest to declare.

scholarly journals PSDX: A Comprehensive Multi-Omics Association Database of Populus trichocarpa With a Focus on the Secondary Growth in Response to Stresses

2021 ◽  
Vol 12 ◽  
Author(s):  
Huiyuan Wang ◽  
Sheng Liu ◽  
Xiufang Dai ◽  
Yongkang Yang ◽  
Yunjun Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Populus Trichocarpa ◽  
Secondary Growth ◽  
A Genome ◽  
Post Transcriptional Regulation

Populus trichocarpa (P. trichocarpa) is a model tree for the investigation of wood formation. In recent years, researchers have generated a large number of high-throughput sequencing data in P. trichocarpa. However, no comprehensive database that provides multi-omics associations for the investigation of secondary growth in response to diverse stresses has been reported. Therefore, we developed a public repository that presents comprehensive measurements of gene expression and post-transcriptional regulation by integrating 144 RNA-Seq, 33 ChIP-seq, and six single-molecule real-time (SMRT) isoform sequencing (Iso-seq) libraries prepared from tissues subjected to different stresses. All the samples from different studies were analyzed to obtain gene expression, co-expression network, and differentially expressed genes (DEG) using unified parameters, which allowed comparison of results from different studies and treatments. In addition to gene expression, we also identified and deposited pre-processed data about alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI). The post-transcriptional regulation, differential expression, and co-expression network datasets were integrated into a new P. trichocarpa Stem Differentiating Xylem (PSDX) database, which further highlights gene families of RNA-binding proteins and stress-related genes. The PSDX also provides tools for data query, visualization, a genome browser, and the BLAST option for sequence-based query. Much of the data is also available for bulk download. The availability of PSDX contributes to the research related to the secondary growth in response to stresses in P. trichocarpa, which will provide new insights that can be useful for the improvement of stress tolerance in woody plants.

scholarly journals Influenza Virus Usurps an Interferon-Induced Translational Program to Promote Viral Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 134
Author(s):  
Mitchell P. Ledwith ◽  
Vy Tran ◽  
Thiprampai Thamamongood ◽  
Christina A. Higgins ◽  
Shashank Tripathi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Viral Replication ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Translational Efficiency ◽  
A Genome ◽  
Cellular Mrnas ◽  
Stimulation Of

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication.

Amplification of 6p Associated with Familial CLL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2432-2432
Author(s):  
Jennifer R Brown ◽  
Bethany Tesar ◽  
Megan Hanna ◽  
Megan Ash ◽  
Stacey M Fernandes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Second Generation ◽  
Somatic Mutations ◽  
Risk Allele ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
High Density ◽  
A Genome

Abstract Abstract 2432 Chronic lymphocytic leukemia (CLL) is one of the most familial of all cancers but the genetic basis of this heritability remains poorly characterized. Families with very strong inheritance of CLL have been described in the literature, and recently the occurrence of CLL in one such family was associated with a polymorphism in the DAPK gene. Here we report the genomic characterization of a family in which CLL appears to be inherited in a Mendelian autosomal dominant manner. Within this family, five of eleven siblings of the first generation were affected, and one of those affected siblings had five children, of whom three were also affected (the second generation). The children of the second generation are currently aged 20–30 and hence too young to know whether they will develop CLL. We performed high-density single-nucleotide polymorphism (SNP) array analysis and gene expression profiling on tumor and germline DNA from four of the offspring of the second generation, as well as six of their children. Analysis of the SNP array data revealed a significant germline amplification of 6p, spanning 0–720 Mb and encompassing a known copy number variant (CNV) region but significantly larger than the CNV region. This amplification was found in both affected individuals with samples available from the second generation, and was transmitted by each of them to one of their two children in the third generation. This amplification was absent from the two unaffected members of the second generation, their children, or any of the other 189 individuals with CLL who were analyzed in our high-density SNP array dataset. None of the unaffected individuals with or without the amplification had evidence of monoclonal B cell lymphocytosis (MBL) by highly sensitive flow cytometry. These unaffected individuals also lacked any PCR-detectable oligoclonal or monoclonal immunoglobulin heavy chain gene rearrangement suggestive of MBL. The region of amplification contains four protein-coding genes: EXOC2, DUSP22, HUS1B and IRF4. We sequenced the coding regions of these four genes and the 5` and 3` UTRs of IRF4 in all family members, but found no somatic mutations in this family. All four genes were also sequenced in 92 other familial CLLs, identifying no somatic mutations. We then analyzed our gene expression profiling data to assess whether any genes in this region were altered in the affected individuals with the amplification. This analysis revealed a significant 1.74X increase in IRF4 expression in the CLLs with the amplification compared to those without (q value < 0.001). By Western blotting, we confirmed that IRF4 protein was increased approximately two-fold in amplified compared to non-amplified samples. These data suggest that the amplification may target IRF4, which has been previously implicated in CLL by a genome wide association study that identified a tag SNP in its 3` UTR as a CLL risk allele. Further analysis of our SNP data demonstrated allele specific amplification in this region, and mass-spectrometric genotyping confirmed enrichment of the CLL risk allele in the individuals with amplification. We conclude that amplification of IRF4 carrying the risk allele for CLL appears likely to be the culprit predisposing to CLL in this family. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The hnRNPs F and H2 bind to similar sequences to influence gene expression

2005 ◽  
Vol 393 (1) ◽  
pp. 361-371 ◽  
Author(s):  
Serkan A. Alkan ◽  
Kathleen Martincic ◽  
Christine Milcarek
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Reporter Expression ◽  
Hnrnp F ◽  
Influence Gene Expression

The hnRNPs (heterogeneous nuclear ribonucleoproteins) F and H2 share a similar protein structure. Both have been implicated as regulating polyadenylation, but hnRNP H2 had a positive effect, whereas hnRNP F acted negatively. We therefore carried out side-by-side comparisons of their RNA-binding and in vivo actions. The binding of the CstF2 (64 kDa cleavage stimulatory factor) to SV40 (simian virus 40) late pre-mRNA substrates containing a downstream GRS (guanine-rich sequence) was reduced by hnRNP F, but not by hnRNP H2, in a UV-cross-linking assay. Point mutations of the 14-nt GRS influenced the binding of purified hnRNP F or H2 in parallel. Co-operative binding of the individual proteins to RNA was lost with mutations of the GRS in the G1−5 or G12−14 regions; both regions seem to be necessary for optimal interactions. Using a reporter green fluorescent protein assay with the GRS inserted downstream of the poly(A) (polyadenine) signal, expression in vivo was diminished by a mutant G1−5 sequence which decreased binding of both hnRNPs (SAA20) and was enhanced by a 12–14-nt mutant that showed enhanced hnRNP F or H2 binding (SAA10). Using small interfering RNA, down-regulation of hnRNP H2 levels diminished reporter expression, confirming that hnRNP H2 confers a positive influence; in contrast, decreasing hnRNP F levels had a negligible influence on reporter expression with the intact GRS. A pronounced diminution in reporter expression was seen with the SAA20 mutant for both. Thus the relative levels of hnRNP F and H2 in cells, as well as the target sequences in the downstream GRS on pre-mRNA, influence gene expression.

scholarly journals Multiplexed functional genomic analysis of 5’ untranslated region mutations across the spectrum of prostate cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yiting Lim ◽  
Sonali Arora ◽  
Samantha L. Schuster ◽  
Lukas Corey ◽  
Matthew Fitzgibbon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Point Mutations ◽  
Genomic Analysis ◽  
Mrna Translation ◽  
Specific Gene ◽  
Functional Genomic Analysis ◽  
A Genome ◽  
Functional Consequences ◽  
Wide Scale

AbstractThe functional consequences of genetic variants within 5’ untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5’ UTR mutations in human prostate cancer. We show that 5’ UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5’ UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5’ UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5’ UTRs are functional in cancer.

scholarly journals Decoding differential gene expression

2020 ◽  
Author(s):  
Shinya Tasaki ◽  
Chris Gaiteri ◽  
Sara Mostafavi ◽  
Yanling Wang
Keyword(s):  
Gene Expression ◽  
Systems Biology ◽  
Differential Gene Expression ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Human Lifespan ◽  
Disease Biology ◽  
A Genome ◽  
Wide Scale ◽  
Differential Gene

AbstractIdentifying the molecular mechanisms that control differential gene expression (DE) is a major goal of basic and disease biology. Combining the strengths of systems biology and deep learning in a model called DEcode, we are able to predict DE more accurately than traditional sequence-based methods, which do not utilize systems biology data. To determine the biological origins of this accuracy, we identify the most predictive regulators and types of regulatory interactions in DEcode, contrasting their roles across many human tissues. Diverse systems biology, ontological and disease-related assessments all point to the predominant influence of post-translational RNA-binding factors on DE. Through the combinatorial gene regulation that is captured in DEcode, it is even possible to predict relatively subtle person-to-person variation in gene expression. We demonstrate the broad applicability of these clinically-relevant predictions by predicting drivers of aging throughout the human lifespan, gene coexpression relationships on a genome-wide scale, and frequent DE in diverse conditions. Researchers can freely access DEcode to utilize genomic big data in identifying influential molecular mechanisms for any human expression data - www.differentialexpression.org.

scholarly journals Adaptive post-transcriptional reprogramming of metabolism limits response to targeted therapy in BRAFV600 melanoma

2019 ◽  
Author(s):  
Lorey K Smith ◽  
Tiffany Parmenter ◽  
Margarete Kleinschmidt ◽  
Eric P Kusnadi ◽  
Jian Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Targeted Therapy ◽  
Rna Binding ◽  
Metabolic Response ◽  
Residual Disease ◽  
Therapy Response ◽  
Metabolic Adaptation ◽  
Drug Induced ◽  
A Genome

AbstractDespite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by a residual disease that results in relapse. This residual disease is characterized by drug-induced adaptation, that in melanoma includes altered metabolism. Here, we examined how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNAi screen and global gene expression profiling. This systematic approach revealed post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA binding kinase UHMK1 interacts with mRNAs that encode metabolic proteins and selectively controls their transport and translation during adaptation to BRAF targeted therapy. Inactivation of UHMK1 improves metabolic response to BRAF targeted therapy and delays resistance to BRAF and MEK combination therapy in vivo. Our data support a model wherein post-transcriptional gene expression pathways regulate metabolic adaptation underpinning targeted therapy response and suggest inactivation of these pathways may delay disease relapse.

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