RESUMEN
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Asunto(s)
Regiones no Traducidas 3'/genética , Evolución Biológica , Enfermedad/genética , Estudio de Asociación del Genoma Completo , Algoritmos , Alelos , Regulación de la Expresión Génica , Genes Reporteros , Variación Genética , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Polirribosomas/metabolismo , Sitios de Carácter Cuantitativo/genética , ARN/genéticaRESUMEN
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Mutación/genética , Motivos de Nucleótidos/genética , Transición de Fase , Mutación Puntual/genética , Poli A/metabolismo , Unión Proteica , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Asunto(s)
ADP-Ribosilación , Neoplasias Ováricas/metabolismo , Biosíntesis de Proteínas , Proteostasis , Ribosomas/metabolismo , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Estrés del Retículo Endoplásmico , Trompas Uterinas/metabolismo , Femenino , Humanos , Ratones Endogámicos NOD , Ratones SCID , NAD/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa , Conformación de Ácido Nucleico , Neoplasias Ováricas/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions (UTRs). The expression of alternative 3' UTR isoforms is highly cell type specific and is further controlled in a gene-specific manner by environmental cues. In this Review, we discuss how the dynamic, fine-grained regulation of APA is accomplished by several mechanisms, including cis-regulatory elements in RNA and DNA and factors that control transcription, pre-mRNA cleavage and post-transcriptional processes. Furthermore, signalling pathways modulate the activity of these factors and integrate APA into gene regulatory programmes. Dysregulation of APA can reprogramme the outcome of signalling pathways and thus can control cellular responses to environmental changes. In addition to the regulation of protein abundance, APA has emerged as a major regulator of mRNA localization and the spatial organization of protein synthesis. This role enables the regulation of protein function through the addition of post-translational modifications or the formation of protein-protein interactions. We further discuss recent transformative advances in single-cell RNA sequencing and CRISPR-Cas technologies, which enable the mapping and functional characterization of alternative 3' UTRs in any biological context. Finally, we discuss new APA-based RNA therapeutics, including compounds that target APA in cancer and therapeutic genome editing of degenerative diseases.
Asunto(s)
Regulación de la Expresión Génica , Poliadenilación , Poliadenilación/genética , Regiones no Traducidas 3'/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica/genética , Biosíntesis de ProteínasRESUMEN
Alternative polyadenylation (APA) is a major driver of transcriptome diversity in human cells. Here, we use deep learning to predict APA from DNA sequence alone. We trained our model (APARENT, APA REgression NeT) on isoform expression data from over 3 million APA reporters. APARENT's predictions are highly accurate when tasked with inferring APA in synthetic and human 3'UTRs. Visualizing features learned across all network layers reveals that APARENT recognizes sequence motifs known to recruit APA regulators, discovers previously unknown sequence determinants of 3' end processing, and integrates these features into a comprehensive, interpretable, cis-regulatory code. We apply APARENT to forward engineer functional polyadenylation signals with precisely defined cleavage position and isoform usage and validate predictions experimentally. Finally, we use APARENT to quantify the impact of genetic variants on APA. Our approach detects pathogenic variants in a wide range of disease contexts, expanding our understanding of the genetic origins of disease.
Asunto(s)
Aprendizaje Profundo , Modelos Genéticos , Poliadenilación/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases/genética , Bases de Datos Genéticas , Expresión Génica/genética , Células HEK293 , Humanos , Mutagénesis/genética , División del ARN/genética , ARN Mensajero/genética , RNA-Seq , Biología Sintética , TranscriptomaRESUMEN
RNA tailing, or the addition of non-templated nucleotides to the 3' end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.
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Regulación de la Expresión Génica/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/fisiología , Animales , Regulación de la Expresión Génica/genética , Humanos , MicroARNs/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Poliadenilación , ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.
Asunto(s)
Poliadenilación , Proteína p53 Supresora de Tumor , Humanos , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Regiones no Traducidas 3'/genética , Regulación de la Expresión Génica , IntronesRESUMEN
Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.
Asunto(s)
Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Interferón gamma/genética , ARN Mensajero/genética , Linfocitos T/inmunología , Animales , Células Cultivadas , Represión Epigenética , Memoria Inmunológica , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Extensión de la Cadena Peptídica de Translación , Ribosomas/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMEN
The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.
Asunto(s)
Poliadenilación , Isoformas de ARN , Isoformas de ARN/genética , Regiones no Traducidas 5' , Regiones no Traducidas 3'/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Exonucleasas/genéticaRESUMEN
Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3' UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3' UTR in proliferating spermatogonia but a short 3' UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3' UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3' UTR versus short 3' UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3' end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.
Asunto(s)
Células Madre Adultas , Isoformas de ARN , Regiones no Traducidas 3'/genética , Células Madre Adultas/metabolismo , Animales , Masculino , Poliadenilación , Isoformas de Proteínas/genética , Isoformas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.
Asunto(s)
Procesamiento Postranscripcional del ARN , Trypanosoma brucei brucei , Glicoproteínas Variantes de Superficie de Trypanosoma , Regiones no Traducidas 3'/genética , Adenosina/análogos & derivados , Regulación de la Expresión Génica , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Neuronas/metabolismo , Empalme Alternativo/genética , Secuencias de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteínas ELAV/química , Larva/metabolismo , Mutación/genética , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
Asunto(s)
Regiones no Traducidas 3'/genética , Antígenos de Neoplasias/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/patología , ARN Mensajero/metabolismo , Ubiquitina/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Animales , Antígenos de Neoplasias/genética , Apoptosis , Biomarcadores de Tumor , Carcinogénesis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Poliadenilación , Empalme del ARN , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA-target interactions by disrupting each predicted miRNA-binding site by CRISPR-Cas9 genome editing in C. elegans We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes. We used this approach to screen for phenotypic targets of the essential mir-35-42 family. By generating 1130 novel 3'UTR alleles across all predicted targets, we identified egl-1 as a phenotypic target whose derepression partially phenocopies the mir-35-42 mutant phenotype by inducing embryonic lethality and low fecundity. These phenotypes can be rescued by compensatory CRISPR mutations that retarget mir-35 to the mutant egl-1 3'UTR. This study demonstrates that the application of in vivo whole organismal CRISPR screening has great potential to accelerate the discovery of phenotypic negative regulatory elements in the noncoding genome.
Asunto(s)
Caenorhabditis elegans/genética , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Alelos , Animales , Sitios de Unión/genética , Sistemas CRISPR-Cas , Edición Génica , Pruebas Genéticas , MicroARNs/genética , Mutación , FenotipoRESUMEN
Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.
Asunto(s)
MicroARNs , Humanos , Regiones no Traducidas 3'/genética , MicroARNs/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Línea Celular , Unión ProteicaRESUMEN
Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA-protein cross-linking with a pulsed femtosecond ultraviolet laser, followed by immunoprecipitation and high-throughput sequencing, allows the determination of binding and dissociation kinetics of the RBP DAZL for thousands of individual RNA-binding sites in cells. This kinetic cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL binds to many RNAs in clusters of multiple proximal sites. The effect of DAZL on mRNA levels and ribosome association correlates with the cumulative probability of DAZL binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects DAZL-RNA binding to DAZL function. Our results show how kinetic parameters for RNA-protein interactions can be measured in cells, and how these data link RBP-RNA binding to the cellular function of RBPs.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión/genética , Línea Celular , Cinética , Rayos Láser , Ratones , Unión Proteica , ARN/genética , Proteínas de Unión al ARN/genética , Ribosomas/metabolismoRESUMEN
Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Leucemia/genética , Complejos Multiproteicos/genética , Receptores CXCR4/genética , Regiones no Traducidas 3'/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/química , Movimiento Celular/genética , Proteínas del Citoesqueleto/genética , Proteína 1 Similar a ELAV/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia/patología , Complejos Multiproteicos/química , Transporte de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.
Asunto(s)
Enfermedad de Huntington , MicroARNs , Humanos , Regiones no Traducidas 3'/genética , Endodesoxirribonucleasas , Exodesoxirribonucleasas/genética , Estudio de Asociación del Genoma Completo , Enfermedad de Huntington/genética , MicroARNs/genética , Enzimas MultifuncionalesRESUMEN
The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.
Asunto(s)
Regiones no Traducidas 3' , Estadios del Ciclo de Vida , Proteínas Protozoarias , Proteínas de Unión al ARN , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Estadios del Ciclo de Vida/genética , Regiones no Traducidas 3'/genética , Animales , Transcriptoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Poli A/metabolismo , Poli A/genética , PoliadenilaciónRESUMEN
Knockout of the ubiquitously expressed miRNA-17â¼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17â¼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17â¼92:Bim interactions to the complex miR-17â¼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17â¼92 seed matches. Blocking miR-17â¼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17â¼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17â¼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.