RESUMEN
Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against Covid-19. During ribosomal translation, a fraction of elongating ribosomes slips by one base in the 5' direction and enters a new reading frame for viral protein synthesis. Any interference with this process profoundly affects viral replication and propagation. For Covid-19, two RNA sites associated with ribosomal frameshifting for SARS-CoV-2 are positioned on the 5' and 3' of the frameshifting residues. Although much attention has been on the 3' frameshift element (FSE), the 5' stem-loop (attenuator hairpin, AH) can play a role. The formation of AH has been suggested to occur as refolding of the 3' RNA structure is triggered by ribosomal unwinding. However, the attenuation activity and the relationship between the two regions are unknown. To gain more insight into these two related viral RNAs and to further enrich our understanding of ribosomal frameshifting for SARS-CoV-2, we explore the RNA folding of both 5' and 3' regions associated with frameshifting. Using our graph-theory-based modeling tools to represent RNA secondary structures, "RAG" (RNA- As-Graphs), and conformational landscapes to analyze length-dependent conformational distributions, we show that AH coexists with the 3-stem pseudoknot of the 3' FSE (graph 3_6 in our dual graph notation) and alternative pseudoknot (graph 3_3) but less likely with other 3' FSE alternative folds (such as 3-way junction 3_5). This is because an alternative length-dependent Stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. In addition, we design four mutants for long lengths that stabilize or disrupt AH, AS1 or FSE pseudoknot to illustrate the deduced AH/AS1 roles and favor the 3_5, 3_6 or stem-loop. These mutants further show how a strengthened pseudoknot can result from a weakened AS1, while a dominant stem-loop occurs with a strengthened AS1. These structural and mutational insights into both ends of the FSE in SARS-CoV-2 advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting a sequence of length-dependent folds, which in turn define potential therapeutic intervention techniques involving both elements. Our work also highlights the complexity of viral landscapes with length-dependent folds, and challenges in analyzing these multiple conformations.
RESUMEN
The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.
RESUMEN
RNA molecules fold to form complex internal structures. Many of these RNA structures populate ensembles with rheostat-like properties, with each state having a distinct function. Until recently, analysis of RNA structures, especially within cells, was limited to modeling either a single averaged structure or computationally-modeled ensembles. These approaches obscure the intrinsic heterogeneity of many structured RNAs. Single-molecule correlated chemical probing (smCCP) strategies are now making it possible to measure and deconvolute RNA structure ensembles based on efficiently executed chemical probing experiments. Here, we provide an overview of fundamental single-molecule probing principles, review current ensemble deconvolution strategies, and discuss recent applications to diverse biological systems. smCCP is enabling a revolution in understanding how the plasticity of RNA structure is exploited in biological systems to respond to stimuli and alter gene function. The energetics of RNA ensembles are often subtle and a subset can likely be targeted to modulate disease-associated biological processes.
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Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.
Asunto(s)
Diferenciación Celular , Proteínas de Drosophila , Células-Madre Neurales , Neuronas , Estabilidad del ARN , ARN Mensajero , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Neuronas/metabolismo , Neuronas/citología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Diferenciación Celular/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Codón/genética , Drosophila melanogaster/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Drosophila/genética , Drosophila/metabolismo , Encéfalo/metabolismo , Encéfalo/citología , Factores de TranscripciónRESUMEN
While many disease-associated single nucleotide polymorphisms (SNPs) are expression quantitative trait loci (eQTLs), a large proportion of genome-wide association study (GWAS) variants are of unknown function. Alternative polyadenylation (APA) plays an important role in posttranscriptional regulation by allowing genes to shorten or extend 3' untranslated regions (UTRs). We hypothesized that genetic variants that affect APA in lung tissue may lend insight into the function of respiratory associated GWAS loci. We generated alternative polyadenylation (apa) QTLs using RNA sequencing and whole genome sequencing on 1241 subjects from the Lung Tissue Research Consortium (LTRC) as part of the NHLBI TOPMed project. We identified 56 179 APA sites corresponding to 13 582 unique genes after filtering out APA sites with low usage. We found that a total of 8831 APA sites were associated with at least one SNP with q-value < 0.05. The genomic distribution of lead APA SNPs indicated that the majority are intronic variants (33%), followed by downstream gene variants (26%), 3' UTR variants (17%), and upstream gene variants (within 1 kb region upstream of transcriptional start site, 10%). APA sites in 193 genes colocalized with GWAS data for at least one phenotype. Genes containing the top APA sites associated with GWAS variants include membrane associated ring-CH-type finger 2 (MARCHF2), nectin cell adhesion molecule 2 (NECTIN2), and butyrophilin subfamily 3 member A2 (BTN3A2). Overall, these findings suggest that APA may be an important mechanism for genetic variants in lung function and chronic obstructive pulmonary disease (COPD).
Asunto(s)
Regiones no Traducidas 3' , Estudio de Asociación del Genoma Completo , Pulmón , Poliadenilación , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Sitios de Carácter Cuantitativo/genética , Humanos , Regiones no Traducidas 3'/genética , Poliadenilación/genética , Pulmón/metabolismo , Masculino , Predisposición Genética a la Enfermedad , Enfermedad Pulmonar Obstructiva Crónica/genética , Femenino , Regulación de la Expresión Génica/genéticaRESUMEN
The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.
RESUMEN
Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent expression in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for expression control and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA and protein expression.
RESUMEN
Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. Here, we explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. We provide evidence for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species we found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. Our findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Histonas/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Homeostasis , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Although not canonically polyadenylated, the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is stabilized by a highly conserved 76-nt triple helix structure on its 3' end. The entire MALAT1 transcript is over 8000 nt long in humans. The strongest structural conservation signal in MALAT1 (as measured by covariation of base pairs) is in the triple helix structure. Primary sequence analysis of covariation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in-cell and cell-free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell-free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing covariation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved, at least in primates and across multiple cell types and conditions.
Asunto(s)
ARN Largo no Codificante , Animales , Humanos , Chlorocebus aethiops , ARN Largo no Codificante/metabolismo , Emparejamiento Base , Línea Celular , Estabilidad del ARN , Proliferación Celular , Línea Celular TumoralRESUMEN
SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5'-untranslated region (5'-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5'-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5'-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5'-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5'-UTR structure. However, those mutations that disrupted the helix that anchors the 5'-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5'-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5'-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.
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Biosíntesis de Proteínas , Enfermedad Pulmonar Obstructiva Crónica , alfa 1-Antitripsina/genética , Regiones no Traducidas 5' , Humanos , Inhibidores de Proteasas , Enfermedad Pulmonar Obstructiva Crónica/genética , ARN Mensajero/metabolismoRESUMEN
Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2'-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.
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Rotavirus , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estructura Secundaria de Proteína , ARN Viral/genética , ARN Viral/metabolismo , Rotavirus/genética , Transcriptoma/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor mRNA (pre-mRNA) structure and downstream function are particularly challenging. Here, we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on pre-mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the Bact complex with pre-mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a ß-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and 6 newly discovered mutations on inclusion of exon 10 of MAPT. This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.
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Empalme Alternativo , Precursores del ARN , Exones , Intrones , Mutación , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , ARN Mensajero/genéticaRESUMEN
Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.
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Drosophila melanogaster , Drosophila , Animales , Codón/genética , Uso de Codones , Drosophila/genética , Drosophila melanogaster/genética , Femenino , Humanos , Masculino , TestículoRESUMEN
Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.
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Proteína 2 Similar a ELAV , Proteínas de Unión al ARN , Tristetraprolina/metabolismo , Regiones no Traducidas 3'/genética , Animales , Ratones , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , RNA-SeqRESUMEN
RNA research is advancing at an ever increasing pace. The newest and most state-of-the-art instruments and techniques have made possible the discoveries of new RNAs, and they have carried the field to new frontiers of disease research, vaccine development, therapeutics, and architectonics. Like proteins, RNAs show a marked relationship between structure and function. A deeper grasp of RNAs requires a finer understanding of their elaborate structures. In pursuit of this, cutting-edge experimental and computational structure-probing techniques output several candidate geometries for a given RNA, each of which is perfectly aligned with experimentally determined parameters. Identifying which structure is the most accurate, however, remains a major obstacle. In recent years, several algorithms have been developed for ranking candidate RNA structures in order from most to least probable, though their levels of accuracy and transparency leave room for improvement. Most recently, advances in both areas are demonstrated by rsRNASP, a novel algorithm proposed by Tan et al. rsRNASP is a residue-separation-based statistical potential for three-dimensional structure evaluation, and it outperforms the leading algorithms in the field.
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Algoritmos , ARN , Conformación de Ácido Nucleico , Proteínas , ARN/química , ARN/genética , Análisis de Secuencia de ARNRESUMEN
Disease-associated variants (DAVs) are commonly considered either through a genomic lens that describes variant function at the DNA level, or at the protein function level if the variant is translated. Although the genomic and proteomic effects of variation are well-characterized, genetic variants disrupting post-transcriptional regulation is another mechanism of disease that remains understudied. Specific RNA sequence motifs mediate post-transcriptional regulation both in the nucleus and cytoplasm of eukaryotic cells, often by binding to RNA-binding proteins or other RNAs. However, many DAVs map far from these motifs, which suggests deeper layers of post-transcriptional mechanistic control. Here, we consider a transcriptomic framework to outline the importance of post-transcriptional regulation as a mechanism of disease-causing single-nucleotide variation in the human genome. We first describe the composition of the human transcriptome and the importance of abundant yet overlooked components such as introns and untranslated regions (UTRs) of messenger RNAs (mRNAs). We present an analysis of Human Gene Mutation Database variants mapping to mRNAs and examine the distribution of causative disease-associated variation across the transcriptome. Although our analysis confirms the importance of post-transcriptional regulatory motifs, a majority of DAVs do not directly map to known regulatory motifs. Therefore, we review evidence that regions outside these well-characterized motifs can regulate function by RNA structure-mediated mechanisms in all four elements of an mRNA: exons, introns, 5' and 3' UTRs. To this end, we review published examples of riboSNitches, which are single-nucleotide variants that result in a change in RNA structure that is causative of the disease phenotype. In this review, we present the current state of knowledge of how DAVs act at the transcriptome level, both through altering post-transcriptional regulatory motifs and by the effects of RNA structure.
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Proteómica , Proteínas de Unión al ARN , Regiones no Traducidas 3' , Variación Genética , Humanos , Nucleótidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.
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Dominio Catalítico , Proteínas Protozoarias/genética , ARN Protozoario/genética , ARN/genética , Telomerasa/genética , Trypanosoma brucei brucei/genética , Secuencia de Bases , Biocatálisis , Pruebas de Enzimas/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , ARN/química , ARN/metabolismo , Pliegue del ARN , ARN Protozoario/química , ARN Protozoario/metabolismo , Telomerasa/química , Telomerasa/metabolismo , Termodinámica , Trypanosoma brucei brucei/metabolismoRESUMEN
SHAPE-JuMP is a concise strategy for identifying close-in-space interactions in RNA molecules. Nucleotides in close three-dimensional proximity are crosslinked with a bi-reactive reagent that covalently links the 2'-hydroxyl groups of the ribose moieties. The identities of crosslinked nucleotides are determined using an engineered reverse transcriptase that jumps across crosslinked sites, resulting in a deletion in the cDNA that is detected using massively parallel sequencing. Here we introduce ShapeJumper, a bioinformatics pipeline to process SHAPE-JuMP sequencing data and to accurately identify through-space interactions, as observed in complex JuMP datasets. ShapeJumper identifies proximal interactions with near-nucleotide resolution using an alignment strategy that is optimized to tolerate the unique non-templated reverse-transcription profile of the engineered crosslink-traversing reverse-transcriptase. JuMP-inspired strategies are now poised to replace adapter-ligation for detecting RNA-RNA interactions in most crosslinking experiments.
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ADN Complementario/química , ARN/química , Programas Informáticos , Algoritmos , Sitios de Unión , Biología Computacional , Reactivos de Enlaces Cruzados , ADN Complementario/genética , Ingeniería Genética , Modelos Moleculares , Conformación de Ácido Nucleico , ARN/genética , Alineación de Secuencia/estadística & datos numéricosRESUMEN
α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.
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Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , alfa 1-Antitripsina/genética , Línea Celular , Codón de Terminación/genética , Regulación de la Expresión Génica/genética , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Pulmón/patología , Macrófagos/metabolismo , Poliadenilación/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , RNA-Seq , Análisis de la Célula Individual , Linfocitos T/metabolismoRESUMEN
Cigarette smoking induces a profound transcriptomic and systemic inflammatory response. Previous studies have focused on gene level differential expression of smoking, but the genome-wide effects of smoking on alternative isoform regulation have not yet been described. We conducted RNA sequencing in whole-blood samples of 454 current and 767 former smokers in the COPDGene Study, and we analyzed the effects of smoking on differential usage of isoforms and exons. At 10% FDR, we detected 3167 differentially expressed genes, 945 differentially used isoforms and 160 differentially used exons. Isoform switch analysis revealed widespread 3' UTR lengthening associated with cigarette smoking. The lengthening of these 3' UTRs was consistent with alternative usage of distal polyadenylation sites, and these extended 3' UTR regions were significantly enriched with functional sequence elements including microRNA and RNA-protein binding sites. These findings warrant further studies on alternative polyadenylation events as potential biomarkers and novel therapeutic targets for smoking-related diseases.