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RNA-binding proteins (RBPs) play critical roles in genome regulation. In this study, we explored the latent function of KPNA2, which is an essential member of the RBP family, in the regulation of alternative splicing (AS) in gastric cancer (GC). We analyzed the role of KPNA2 in regulating differential expression and AS via RNA sequencing (RNA-seq) and improved RNA immunoprecipitation sequencing (iRIP-seq). Clinical specimens were used to analyze the associations between KPNA2 expression and clinicopathological characteristics. CCK8 assays, transwell assays and wound healing assays were performed to explore the effect of KPNA2/WDR62 on GC cell progression. KPNA2 was shown to be highly expressed in GC cells and tissues and associated with lymph node metastases. KPNA2 promoted the proliferation, migration and invasion of GC cells and primarily regulated exon skipping, alternative 3's splice sites (A3SSs), alternative 5' splice sites (A5SSs), and cassette exons. We further revealed that KPNA2 participated in biological processes related to cell proliferation, and the immune response in GC via the regulation of transcription. In addition, KPNA2 preferentially bound to intron regions. Notably, KPNA2 regulated the A3SS AS mode of WDR62, and upregulation of WDR62 reversed the KPNA2 downregulation-induced inhibition of GC cell proliferation, migration and invasion. Finally, we discovered that the AS of immune-related molecules could be regulated by KPNA2. Overall, our results demonstrated for the first time that KPNA2 functions as an oncogenic splicing factor in GC that regulated the AS and differential expression of GC-related genes, and KPNA2 may be a potential target for GC treatment.

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Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

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We present a large, ten-generation family of 273 individuals with 84 people having preaxial polydactyly/triphalangeal thumb due to a pathogenic variant in the zone of polarizing activity regulatory sequence (ZRS) within the exon 5 of LMBR1. The causative change maps to position 396 of the ZRS, located at position c.423 + 4909C > T (chr7:156791480; hg38; LMBR1 ENST00000353442.10; rs606231153 NG_009240.2) in the intron 5 of LMBR1. The first affected individual with the disorder was traced back to mid-1700, when some settlers and workers established in Cervera de Buitrago, a small village about 82 km North to Madrid. Clinical and radiological studies of most of the affected members have been performed for 42 years (follow-up of the family by LFGA). Molecular studies have confirmed a pathogenic variant in the ZRS that segregates in this family. To the best of our knowledge, this is the largest family with preaxial polydactyly/triphalangeal thumb reported so far.

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Nidovirales is one order of RNA virus, with the largest single-stranded positive sense RNA genome enwrapped with membrane envelope. It comprises four families (Arterividae, Mesoniviridae, Roniviridae, and Coronaviridae) and has been circulating in humans and animals for almost one century, posing great threat to livestock and poultry，as well as to public health. Nidovirales shares similar life cycle: attachment to cell surface, entry, primary translation of replicases, viral RNA replication in cytoplasm, translation of viral proteins, virion assembly, budding, and release. The viral RNA synthesis is the critical step during infection, including genomic RNA (gRNA) replication and subgenomic mRNAs (sg mRNAs) transcription. gRNA replication requires the synthesis of a negative sense full-length RNA intermediate, while the sg mRNAs transcription involves the synthesis of a nested set of negative sense subgenomic intermediates by a discontinuous strategy. This RNA synthesis process is mediated by the viral replication/transcription complex (RTC), which consists of several enzymatic replicases derived from the polyprotein 1a and polyprotein 1ab and several cellular proteins. These replicases and host factors represent the optimal potential therapeutic targets. Hereby, we summarize the Nidovirales classification, associated diseases, "replication organelle," replication and transcription mechanisms, as well as related regulatory factors.

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In a minority of flowering plants, separate sexes are genetically determined by sex chromosomes. The Y chromosome has a non-recombining region that degenerates, causing a reduced expression of Y genes. In some species, the lower Y expression is accompanied by dosage compensation (DC), a mechanism that re-equalizes male and female expression and/or brings XY male expression back to its ancestral level. Here, we review work on DC in plants, which started as early as the late 1960s with cytological approaches. The use of transcriptomics fired a controversy as to whether DC existed in plants. Further work revealed that various plants exhibit partial DC, including a few species with young and homomorphic sex chromosomes. We are starting to understand the mechanisms responsible for DC in some plants, but in most species, we lack the data to differentiate between global and gene-by-gene DC. Also, it is unknown why some species evolve many dosage compensated genes while others do not. Finally, the forces that drive DC evolution remain mysterious, both in plants and animals. We review the multiple evolutionary theories that have been proposed to explain DC patterns in eukaryotes with XY or ZW sex chromosomes. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

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Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3'UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.

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To investigate the role of PRRSV nonstructural proteins (nsps) in viral RNA replication and transcription, we generated a cDNA clone of PRRSV strain NCV1 carrying the nanoluciferase (nluc) gene under the control of the transcription regulatory sequence 6 (TRS6) designated as pNCV1-Nluc. Cells transfected with the pNCV1-Nluc DNA plasmid produced an infectious virus and high levels of luciferase activity. Interestingly, cells transfected with mutant pNCV1-Nluc constructs carrying deletions in nsp7 or nsp9 regions also exhibited luciferase activity, although no infectious virus was produced. Further investigation revealed that the cDNA sequences corresponding to the PRRSV 5' untranslated region (UTR) and TRS, when cloned upstream of the reporter gene nluc, were able to drive the expression of the reporter genes in the transfected cells. Luciferase signals from cells transfected with a reporter plasmid carrying PRRSV 5' UTR or TRS sequences upstream of nluc were in the range of 6- to 10-fold higher compared to cells transfected with an empty plasmid carrying nluc only. The results suggest that PRRSV 5' UTR and TRS-B in their cDNA forms possess cryptic eukaryotic promoter activity.

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Primary σ70 factors are key conserved bacterial regulatory proteins that interact with regulatory DNA to control gene expression. It is, however, poorly understood whether σ70 sequence diversity in different bacteria reflects functional differences. Here, we employ comparative and functional genomics to explore the sequence and function relationship of primary σ70. Using multiplex automated genome engineering and deep sequencing (MAGE-seq), we generate a saturation mutagenesis library and high-resolution fitness map of E. coli σ70 in domains 2-4. Mapping natural σ70 sequence diversity to the E. coli σ70 fitness landscape reveals significant predicted fitness deficits across σ70 orthologs. Interestingly, these predicted deficits are larger than observed fitness changes for 15 σ70 orthologs introduced into E. coli. Finally, we use a multiplexed transcriptional reporter assay and RNA sequencing (RNA-seq) to explore functional differences of several σ70 orthologs. This work provides an in-depth analysis of σ70 sequence and function to improve efforts to understand the evolution and engineering potential of this global regulator.

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Despite the importance of gene regulatory enhancers in human biology and evolution, we lack a comprehensive model of enhancer evolution and function. This substantially limits our understanding of the genetic basis of species divergence and our ability to interpret the effects of noncoding variants on human traits. To explore enhancer sequence evolution and its relationship to regulatory function, we traced the evolutionary origins of transcribed human enhancer sequences with activity across diverse tissues and cellular contexts from the FANTOM5 consortium. The transcribed enhancers are enriched for sequences of a single evolutionary age ("simple" evolutionary architectures) compared with enhancers that are composites of sequences of multiple evolutionary ages ("complex" evolutionary architectures), likely indicating constraint against genomic rearrangements. Complex enhancers are older, more pleiotropic, and more active across species than simple enhancers. Genetic variants within complex enhancers are also less likely to associate with human traits and biochemical activity. Transposable-element-derived sequences (TEDS) have made diverse contributions to enhancers of both architectures; the majority of TEDS are found in enhancers with simple architectures, while a minority have remodeled older sequences to create complex architectures. Finally, we compare the evolutionary architectures of transcribed enhancers with histone-mark-defined enhancers. Our results reveal that most human transcribed enhancers are ancient sequences of a single age, and thus the evolution of most human enhancers was not driven by increases in evolutionary complexity over time. Our analyses further suggest that considering enhancer evolutionary histories provides context that can aid interpretation of the effects of variants on enhancer function. Based on these results, we propose a framework for analyzing enhancer evolutionary architecture.

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In this paper we consider the time evolution of a population of size N with overlapping generations, in the vicinity of m genes. We assume that this population is subject to point mutations, genetic drift, and selection. More specifically, we analyze the statistical distribution of the waiting time Tm until the expression of these genes have changed for all individuals, when transcription factors recognize and attach to short DNA-sequences (binding sites) within regulatory sequences in the neighborhoods of the m genes. The evolutionary dynamics is described by a multitype Moran process, where each individual is assigned a m×L regulatory array that consists of regulatory sequences with L nucleotides for all m genes. We study how the waiting time distribution depends on the number of genes, the mutation rate, the length of the binding sites, the length of the regulatory sequences, and the way in which the targeted binding sites are coordinated for different genes in terms of selection coefficients. These selection coefficients depend on how many binding sites have appeared so far, and possibly on their order of appearance. We also allow for back mutations, whereby some acquired binding sites may be lost over time. It is further assumed that the mutation rate is small enough to warrant a fixed state population, so that all individuals have the same regulatory array, at any given time point, until the next successful mutation arrives in some individual and spreads to the rest of the population. We further incorporate stochastic tunneling, whereby successful mutations get mutated before their fixation. A crucial part of our approach is to divide the huge state space of regulatory arrays into a small number of components, assuming that the array component varies as a Markov process over time. This implies that Tm is the time until this Markov process hits an absorbing state, with a phase-type distribution. A number of interesting results can be derived from our general setup, for instance that the expected waiting time increases exponentially with m, for a selectively neutral model, when back-mutations are possible.

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In the present study we conducted a genome-wide association study (GWAS) in a cohort of 505 patients with paranoid schizophrenia (SCZ), of which 95 had tardive dyskinesia (TD), and 503 healthy controls. Using data generated by the PsychENCODE Consortium (PEC) and other bioinformatic databases, we revealed a gene network, implicated in neurodevelopment and brain function, associated with both these disorders. Almost all these genes are in gene or isoform co-expression PEC network modules important for the functioning of the brain; the activity of these networks is also altered in SCZ, bipolar disorder and autism spectrum disorders. The associated PEC network modules are enriched for gene ontology terms relevant to the brain development and function (CNS development, neuron development, axon ensheathment, synapse, synaptic vesicle cycle, and signaling receptor activity) and to the immune system (inflammatory response). Results of the present study suggest that orofacial and limbtruncal types of TD seem to share the molecular network with SCZ. Paranoid SCZ and abnormal involuntary movements that indicate the orofacial type of TD are associated with the same genomic loci on chromosomes 3p22.2, 8q21.13, and 13q14.2. The limbtruncal type of TD is associated with a locus on chromosome 3p13 where the best functional candidate is FOXP1, a high-confidence SCZ gene. The results of this study shed light on common pathogenic mechanisms for SCZ and TD, and indicate that the pathogenesis of the orofacial and limbtruncal types of TD might be driven by interacting genes implicated in neurodevelopment.

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Reporter gene assays allow for examining the influence of regulatory DNA sequences on the transcription of target genes. In Ewing sarcoma, the study of these DNA sequences is especially paramount for its main driver mutation is a fusion transcription factor that binds different motifs than its wild-type constituents. Here, we describe the process of analyzing the enhancer activity of regulatory DNA sequences using transfection-based dual-luciferase reporter assays in Ewing sarcoma cell lines. To this end, we provide a protocol for cloning sequences of interest from genomic DNA into a firefly luciferase-containing plasmid, transfecting Ewing sarcoma cells with plasmids and measuring luciferase expression by luminescence. The entire procedure can be completed in 14 days.

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Diverse populations worldwide are differentially affected by coronavirus disease 2019 (COVID-19). While socioeconomic background has been studied extensively, little is known about the genetic variation underlying this phenomenon. This study is aimed at examining the genetic basis behind the great discrepancies among diverse ethnic groups in terms of COVID-19 susceptibility for viral infection, disease prognosis, and mortality. To this end, in silico analysis of single-nucleotide polymorphisms (SNPs) within regulatory sequences of the human angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2)-the virus's gateway to host cells-and their plausible implications on expression levels was conducted. We provide indication that the variation in the human ACE2 and TMPRSS2 regulatory sequences is likely to be involved in and contribute to this phenomenon. SNPs that are abundant in the more susceptible populations introduce binding sites (BSs) for transcription factors or they may invalidate BSs for transcription repressor-both may enhance target gene (ACE2 or TMPRSS2) expression in the relevant target tissues. SNPs that are abundant in the more resistant populations may invalidate BSs for a transcriptional repressor or they may introduce BSs for a transcriptional repressor or initiator of mRNA degradation, which may reduce target gene expression levels. This aspect, when added to the socioeconomic factors, can be a cause for the divergent prevalence of the disease and the different mortality rates within diverse populations. This demonstration may call for a shift in the paradigm of searching for COVID-19 biomarkers, such that SNPs within regulatory sequences should be of high importance.

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BACKGROUND: The wide distribution of Aedes aegypti, the main vector of dengue and yellow fever viruses, currently puts three billion people in the world at risk of infection with these viruses. Continuous transmission of these and other viruses despite aggressive efforts to prevent this emphasizes the need to develop new control strategies. Proposals to control disease transmission based on vector engineering, including both population suppression and population replacement, rely on the development of transgenes under the control of regulatory elements able to drive molecules in a specific tissue, time and strength. METHODS: Here we report the characterization of a promoter active in both the female germline and early zygote, derived from the transcription factor bZip1 in the mosquito Ae. aegypti, using transposon-based methods and RT-qPCR. RESULTS: We generated seven transgenic lines carrying AabZip1-reporter constructs and observed expression in both the ovary and early embryo. RT-qPCR analysis was performed to evaluate transcript expression patterns for each line, confirming that transgenic expression from the AabZip1 promoter largely recapitulated the endogenous expression pattern, albeit the strength of maternal expression appeared to be strongly influenced by chromosomal position. CONCLUSIONS: This study provides a new regulatory sequence that can be useful for generating transgenic lines that can become a tool in vector control strategies.

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Gene regulatory regions contain short and degenerated DNA binding sites recognized by transcription factors (TFBS). When TFBS harbor SNPs, the DNA binding site may be affected, thereby altering the transcriptional regulation of the target genes. Such regulatory SNPs have been implicated as causal variants in Genome-Wide Association Study (GWAS) studies. In this study, we describe improved versions of the programs Variation-tools designed to predict regulatory variants, and present four case studies to illustrate their usage and applications. In brief, Variation-tools facilitate i) obtaining variation information, ii) interconversion of variation file formats, iii) retrieval of sequences surrounding variants, and iv) calculating the change on predicted transcription factor affinity scores between alleles, using motif scanning approaches. Notably, the tools support the analysis of haplotypes. The tools are included within the well-maintained suite Regulatory Sequence Analysis Tools (RSAT, http://rsat.eu), and accessible through a web interface that currently enables analysis of five metazoa and ten plant genomes. Variation-tools can also be used in command-line with any locally-installed Ensembl genome. Users can input personal collections of variants and motifs, providing flexibility in the analysis.

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An oncolytic herpes simplex virus (oHSV) has proven amenable in oncolytic virotherapy and was approved to treat melanoma. The immediate-early (IE) protein ICP27 encoded by gene UL54 is essential for HSV infection. Post-transcriptional modification of UL54 would increase tumor targeting of oHSVs. However, UL54 gene transcription regulatory sequences and factors were not reported yet. Here we isolated a new strain LXMW of type 1 HSV (HSV-1-LXMW) in China and found it's closely related to HSV-1 strains Patton and H129 in the US by the first and next generation DNA sequencing viral DNA phylogenetic analysis. Using a weight matrix-based program Match, we found the UL54 transcription regulatory sequences binding to the transcription factors Oct-1, v-Myb and Pax-6 in HSV-1-LXMW, while the sequences binding to Oct-1 and Hairy in a HSV-2 strain. Further validation showed that HSV-1 and HSV-2 shared the common sequence binding to Oct-1, but had unique sequences to bind v-Myb and Pax-6, or Hairy, respectively, by DNA sequence alignment of total 11 HSV strains. The published results howed that the expression of transcription factors is consistent with the tissue tropism of HSV-1 and HSV-2. In the current article a new HSV-1 strain LXMW was isolated and its putative HSV UL54 transcription regulatory sequences and factors were identified for the first time. Our findings highlight the new understanding of the principles of transcriptional regulation in HSV biology and oncolytic virotherapy.

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Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.

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Mechanical biosensors can be used to quantitatively explore DNA-protein binding mechanisms by detecting targets at low concentrations or measuring force in single-molecule force spectroscopy. However, restrictions in single-molecule manipulation and labelling protocols have hindered the application for bulk analysis of label-free protein detection. Here, we present the integration of molecular force measurement and finely tunable detection of label-free proteins into one mechanical sensor. Regulatory-sequence force spectroscopy was obtained to investigate the binding force of DNA G-quadruplexes (GQ) and label-free protein. The dual control of regulatory sequences and mechanical forces induces the structure switching from DNA duplex to GQ/protein complex. It exhibits a synergistic effect, enabling the rational fine-tuning of the dynamic range for biosensing protein concentrations over eight orders of magnitude. Furthermore, this method was exploited to estimate the stability of the human telomeric DNA GQ by Ku protein and ligand methylpyridostatin. The results revealed that human telomeric GQ has two different binding sites for Ku protein and ligand. Force spectroscopy integrating label-free force measurement and tunable target detection holds great promise for use in biosensing, drug screening, targeted therapies, DNA nanotechnology, and fields in which GQ are of rapidly increasing importance.

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Objective@#To detect potential mutation in a Chinese pedigree affected with congenital limb malformations.@*Methods@#Clinical data was collected. Genomic DNA was extracted from peripheral blood samples of family members. The zone of polarizing activity regulatory sequence (ZRS) were amplified by PCR and subjected to direct sequencing.@*Results@#Among the 13 individuals in this pedigree, there were 4 PPD patients, who were characterized by varying degrees of deformity. The female patients suffered triphalangeal thumb and preaxial polydactyly, while the male patients only had preaxial polydactyly. Only one patient had foot involvement. TA heterogeneous mutations was discovered in the ZRS (105C>G) in all patients, the same mutation was not detected in 2 healthy family members.@*Conclusion@#The inheritance pattern of PPD was autosomal dominant inheritance. There was a significant variability of symptoms among family patients. The heterozygous mutation of the ZRS (105C>G) probably underlie the disease.

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Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3' genomic region sites, between ORF4' and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2', TRS4' or TRS7. M013 insertion at the ORF4'/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2' retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2' at the ORF4'/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4'/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

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