A Comprehensive RNA-seq Analysis of Human Bocavirus 1 Transcripts in Infected Human Airway Epithelium

Human bocavirus 1 (HBoV1) infects well-differentiated (polarized) human airway epithelium (HAE) cultured at an air-liquid interface (ALI). In the present study, we applied next-generation RNA sequencing to investigate the genome-wide transcription profile of HBoV1, including viral mRNA and small RNA transcripts, in HBoV1-infected HAE cells. We identified novel transcription start and termination sites and confirmed the previously identified splicing events. Importantly, an additional proximal polyadenylation site (pA)p2 and a new distal polyadenylation site (pA)dREH lying on the right-hand hairpin (REH) of the HBoV1 genome were identified in processing viral pre-mRNA. Of note, all viral nonstructural proteins-encoding mRNA transcripts use both the proximal polyadenylation sites [(pA)p1 and (pA)p2] and distal polyadenylation sites [(pA)d1 and (pA)dREH] for termination. However, capsid proteins-encoding transcripts only use the distal polyadenylation sites. While the (pA)p1 and (pA)p2 sites were utilized at roughly equal efficiency for proximal polyadenylation of HBoV1 mRNA transcripts, the (pA)d1 site was more preferred for distal polyadenylation. Additionally, small RNA-seq analysis confirmed there is only one viral noncoding RNA (BocaSR) transcribed from nt 5199–5340 of the HBoV1 genome. Thus, our study provides a systematic and unbiased transcription profile, including both mRNA and small RNA transcripts, of HBoV1 in HBoV1-infected HAE-ALI cultures.

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Alternative Polyadenylation of Human Bocavirus at Its 3′ End Is Regulated by Multiple Elements and Affects Capsid Expression

Journal of Virology ◽

10.1128/jvi.02026-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 8

Author(s):

Sujuan Hao ◽

Junmei Zhang ◽

Zhen Chen ◽

Huanzhou Xu ◽

Hanzhong Wang ◽

...

Keyword(s):

Nonstructural Protein ◽

Infectious Clone ◽

Alternative Polyadenylation ◽

Polyadenylation Site ◽

Human Bocavirus ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

Mrna Transcripts ◽

The Right

ABSTRACT Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3′ end regulates its capsid expression. IMPORTANCE The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2 does not contain typical polyadenylation signals, and the last 42 nt form a stem-loop structure which is almost identical to that of MVC. Further study showed that NS1, REH, and cis elements of (pA)d are required for efficient polyadenylation at (pA)d2. Polyadenylation at (pA)d2 enhances capsid expression. Our study demonstrates alternative polyadenylation at the 3′ end of HBoV and suggests an additional mechanism by which capsid expression is regulated.

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Benchmarking sequencing methods and tools that facilitate the study of alternative polyadenylation

Genome Biology ◽

10.1186/s13059-021-02502-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Ankeeta Shah ◽

Briana E. Mittleman ◽

Yoav Gilad ◽

Yang I. Li

Keyword(s):

Single Molecule ◽

Alternative Polyadenylation ◽

Regulatory Elements ◽

Polyadenylation Site ◽

Rna Seq ◽

Computational Tools ◽

Protein Coding ◽

Short Read ◽

Processing Event ◽

Polyadenylation Sites

Abstract Background Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3′ ends. Most APA occurs within 3′ UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization. Results APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools—TAPAS, QAPA, DaPars2, GETUTR, and APATrap— against 3′-Seq, a specialized RNA-seq protocol that enriches for reads at the 3′ ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3′-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3′-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL). Conclusions We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3′-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.

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Human bocavirus 1 infects commercially available primary human airway epithelium cultures productively

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.10.012 ◽

2014 ◽

Vol 195 ◽

pp. 112-119 ◽

Cited By ~ 37

Author(s):

Xuefeng Deng ◽

Yi Li ◽

Jianming Qiu

Keyword(s):

Airway Epithelium ◽

Human Bocavirus ◽

Human Airway ◽

Human Airway Epithelium

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JAK inhibitors dampen activation of interferon-stimulated transcription of ACE2 isoforms in human airway epithelial cells

Communications Biology ◽

10.1038/s42003-021-02167-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Hye Kyung Lee ◽

Olive Jung ◽

Lothar Hennighausen

Keyword(s):

Airway Epithelium ◽

Airway Epithelial Cells ◽

Janus Kinase ◽

Cell Tropism ◽

Airway Epithelial ◽

Rna Seq ◽

Jak Inhibitors ◽

Angiotensin Converting Enzyme 2 ◽

Human Airway ◽

Human Airway Epithelium

AbstractSARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE2 and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib, used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.

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Human Bocavirus 1 Infection of Well‐Differentiated Human Airway Epithelium

Current Protocols in Microbiology ◽

10.1002/cpmc.107 ◽

2020 ◽

Vol 58 (1) ◽

Cited By ~ 1

Author(s):

Ziying Yan ◽

Xuefeng Deng ◽

Jianming Qiu

Keyword(s):

Airway Epithelium ◽

Human Bocavirus ◽

Human Airway ◽

Human Airway Epithelium ◽

Well Differentiated

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Aptardi predicts polyadenylation sites in sample-specific transcriptomes using high-throughput RNA sequencing and DNA sequence

Nature Communications ◽

10.1038/s41467-021-21894-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ryan Lusk ◽

Evan Stene ◽

Farnoush Banaei-Kashani ◽

Boris Tabakoff ◽

Katerina Kechris ◽

...

Keyword(s):

Rna Sequencing ◽

Dna Sequence ◽

Mammalian Species ◽

Alternative Polyadenylation ◽

Sequence Information ◽

Rna Seq ◽

Average Precision ◽

Polyadenylation Sites ◽

Dna Nucleotide Sequence

AbstractAnnotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3′-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model—trained using the Human Brain Reference RNA commercial standard—performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi’s input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

Scientific Reports ◽

10.1038/s41598-021-94782-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nadzeya Marozkina ◽

Laura Smith ◽

Yi Zhao ◽

Joe Zein ◽

James F. Chmiel ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Nitrogen Oxides ◽

Airway Epithelium ◽

Airflow Obstruction ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Airway ◽

Human Airway Epithelium ◽

Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

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