Untranslated Sequence Research Articles

MRNA expression profiles in muscle-derived extracellular vesicles of Large White and wild boar piglets reveal their potential roles in immunity and muscle phenotype.

Extracellular vesicles (EVs) known for their pivotal role in intercellular communication through RNA delivery, hold paramount implications for understanding muscle phenotypic variations in diverse pig breeds. In this study, we compared the mRNA expression profiles of longissimus dorsi muscles and muscle-derived extracellular vesicles (M-EVs), and also examined the diversity of enriched genes in M-EVs between weaned wild boars and commercial Large White pigs with respect to their numbers and biological functions. The results of the study showed that the variation in the expression profiles of mRNAs between muscles and M-EVs was much greater than the variability between the respective breeds. Meanwhile, the enrichment trend of low-expressed genes (ranked <1,000) was significantly (p-value ≤ 0.05) powerful in M-EVs compared to highly expressed genes in muscles. In addition, M-EVs carried a smaller proportion of coding sequences and a larger proportion of untranslated region sequences compared to muscles. There were 2,110 genes enriched in M-EVs (MEGs) in Large White pigs and 2,322 MEGs in wild boars, with 1,490 MEGs shared interbreeds including cyclin D2 (CCND2), which inhibits myogenic differentiation. Of the 89 KEGG pathways that were significantly enriched (p-value ≤ 0.05) for these MEGs, 13 unique to Large White pigs were mainly related to immunity, 27 unique to wild boars were functionally diverse but included cell fate regulation such as the Notch signaling pathway and the TGF-beta signaling pathway, and 49 were common to both breeds were also functionally complex but partially related to innate immunity, such as the Complement and coagulation cascades and the Fc gamma R-mediated phagocytosis. These findings suggest that mRNAs in M-EVs have the potential to serve as indicators of muscle phenotype differences between the two pig breeds, highlighting the need for further exploration into the role of EV-RNAs in pig phenotype formation.

Efficient Production of Enterovirus 71 (EV71) Virus-like Particles by Controlling Promoter Strength in Insect Cells.

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

MiR-128-3p Regulates Follicular Granulosa Cell Proliferation and Apoptosis by Targeting the Growth Hormone Secretagogue Receptor.

The proliferation and apoptosis of granulosa cells (GCs) affect follicle development and reproductive disorders, with microRNAs playing a crucial regulatory role. Previous studies have shown the differential expression of miR-128-3p at different stages of goat follicle development, which suggests its potential regulatory role in follicle development. In this study, through the Cell Counting Kit-8 assay, the EDU assay, flow cytometry, quantitative real-time polymerase chain reaction, Western blot, and the dual-luciferase reporter assay, we used immortal human ovarian granulosa tumor cell line (KGN) cells as materials to investigate the effects of miR-128-3p and its predicted target gene growth hormone secretagogue receptor (GHSR) on GC proliferation and apoptosis. The results show that overexpression of miR-128-3p inhibited the proliferation of KGN cells, promoted cell apoptosis, and suppressed the expression of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2 (BCL2) while promoting that of Bcl-2 associated X protein (BAX). The dual-luciferase reporter assay revealed that miR-128-3p bound to the 3' untranslated region sequence of GHSR, which resulted in the inhibited expression of GHSR protein. Investigation of the effects of GHSR on GC proliferation and apoptosis revealed that GHSR overexpression promoted the expression of PCNA and BCL2, enhanced GC proliferation, and inhibited cell apoptosis, whereas the opposite effects were observed when GHSR expression was inhibited. In addition, miR-128-3p and GHSR can influence the expression of extracellular signal-regulated kinase 1/2 protein. In conclusion, miR-128-3p inhibits KGN cell proliferation and promotes cell apoptosis by downregulating the expression of the GHSR gene.

Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages.

Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

Untranslated Region Sequences and the Efficacy of mRNA Vaccines against Tuberculosis.

mRNA vaccines have been shown to be effective in combating the COVID-19 pandemic. The amount of research on the use of mRNAs as preventive and therapeutic modalities has undergone explosive growth in the last few years. Nonetheless, the issue of the stability of mRNA molecules and their translation efficiency remains incompletely resolved. These characteristics of mRNA directly affect the expression level of a desired protein. Regulatory elements of RNA-5' and 3' untranslated regions (UTRs)-are responsible for translation efficiency. An optimal combination of the regulatory sequences allows mRNA to significantly increase the target protein's expression. We assessed the translation efficiency of mRNA encoding of firefly luciferase with various 5' and 3'UTRs in vitro on cell lines DC2.4 and THP1. We found that mRNAs containing 5'UTR sequences from eukaryotic genes HBB, HSPA1A, Rabb, or H4C2, or from the adenoviral leader sequence TPL, resulted in higher levels of luciferase bioluminescence 4 h after transfection of DC2.4 cells as compared with 5'UTR sequences used in vaccines mRNA-1273 and BNT162b2 from Moderna and BioNTech. mRNA containing TPL as the 5'UTR also showed higher efficiency (as compared with the 5'UTR from Moderna) at generating a T-cell response in mice immunized with mRNA vaccines encoding a multiepitope antigen. By contrast, no effects of various 5'UTRs and 3'UTRs were detectable in THP1 cells, suggesting that the observed effects are cell type specific. Further analyses enabled us to identify potential cell type-specific RNA-binding proteins that differ in landing sites within mRNAs with various 5'UTRs and 3'UTRs. Taken together, our data indicate high translation efficiency of TPL as a 5'UTR, according to experiments on DC2.4 cells and C57BL/6 mice.

Intragenomic rearrangements involving 5′-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses

BackgroundVariation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses that was first documented in an infectious isolate of the betacoronavirus SARS-CoV-2, obtained from 3 patients in Hong Kong that had a 5′-untranslated region segment at the end of the ORF6 gene that in its new location translated into an ORF6 protein with a predicted modified carboxyl terminus. While comparing the amino acid sequences of translated ORF8 genes in the GenBank database, we found a subsegment of the same 5′-UTR-derived amino acid sequence modifying the distal end of ORF8 of an isolate from the United States and decided to carry out a systematic search.MethodsUsing the nucleotide and in the case of SARS-CoV-2 also the translated amino acid sequence in three reading frames of the genomic termini of coronaviruses as query sequences, we searched for 5′-UTR sequences in regions other than the 5′-UTR in SARS-CoV-2 and reference strains of alpha-, beta-, gamma-, and delta-coronaviruses.ResultsWe here report numerous genomic insertions of 5′-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, but not delta- or gammacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions would change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain inserted 5′-UTR sequences.ConclusionThe intragenomic rearrangements involving 5′-untranslated region sequences described here, which in several cases affect highly conserved genes with a low propensity for recombination, may underlie the generation of variants homotypic with those of concern or interest and with potentially differing pathogenic profiles. Intragenomic rearrangements thus add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

Mechanism by Which Inflammation and Oxidative Stress Induce Mineralocorticoid Receptor Gene Expression in Aging Vascular Smooth Muscle Cells.

Vascular MR (mineralocorticoid receptor) expression increases with age driving aging-associated vascular stiffness and hypertension. MR has two isoforms (1α and 1β) with distinct 5'-untranslated and promoter sequences (P1 and P2), but the gene regulatory mechanisms remain unknown. We investigated mechanisms driving MR gene transcriptional regulation in aging human smooth muscle cells (SMC). MR was quantified in aortic tissue and primary human aortic SMC (HASMC) comparing adult and aged donors and adult HASMC treated with H2O2, to induce aging. Predicted transcription factor (TF) binding sites in the MR gene were validated using chromatin immunoprecipitations and reporter assays. The impact of TF inhibitors on MR isoforms and fibrosis target gene expression was examined. Expression of both MR mRNA isoforms increased with donor age or H2O2 treatment in HASMCs. HIF1α (hypoxia-inducible factor) and the inflammatory TF NFκB (nuclear factor kappa B) both increased with age in HASMCs and are predicted to bind MR promoters. H2O2 induced HIF1α and NFκB expression and DNA binding of HIF1α to the MR P1 promoter and of NFκB to both MR promoters in HASMCs. HIF1α inhibition decreased MR-1α isoform expression while NFκB inhibition decreased both MR isoforms. HIF1α, NFκB, and MR inhibition decreased the expression of a SMC-MR target gene implicated in vascular fibrosis. In human aortic tissues, expression of HIF1α and NFκB each positively correlated with donor age and MR expression (P<0.0001). These data implicate the inflammatory TF, NFκB, and oxidative stress-induced TF, HIF1α, in regulating SMC MR transcription in aging HASMCs, which drives aging-related vascular stiffness and cardiovascular disease.

Molecular Evolution of Metallothioneins of Antarctic Fish: A Physiological Adaptation to Peculiar Seawater Chemical Characteristics

Metallothioneins (MTs) are low-molecular weight sulfur-rich proteins, widely distributed in nature. They play a homeostatic role in the control and detoxification of metal ions. Previous studies indicated that MTs also have the capacity to scavenge reactive oxygen species. This study aimed to investigate the evolution of the protein in the notothenioid fish, evolved under the selective pressure of relatively high oxygen partial pressures, characteristics of cold Antarctic seawaters, and relatively high concentrations of metal ions, Cd and Cu in particular. The cDNA sequences of MT isoforms were characterized in members of the Nototheniidae, Bathydraconidae, Artedidraconidae, and Channichthyidae families. The phylogenetic relationships of MTs from these families and other teleosts were inferred by using Maximum Likelihood and Bayesian methods. The analysis of coding region and untranslated (UTR) sequences indicated the presence of two MT clades, each containing one of the two MT isoforms, MT-1 and MT-2. Our results indicated, for the first time for these proteins, that the evolution of MT genes has been characterized by strong purifying selection, whereas it did not observe any evidence of positive selection. In addition, phylogenetic analysis of the UTRs suggested that functional changes, in particular related to the MT-1 gene expression, had accompanied the duplication event.

Synthesis and evaluation of 2,9-disubstituted-1,10-phenanthroline derivatives as G-quadruplex binders

G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences.We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF).

Evidence that untranslated genomic sequences are key determinants of insect-specific flavivirus host restriction

Most flaviviruses cycle between arthropods and vertebrates. Others, such as Long Pine Key virus (LPKV), are insect-specific. We investigated whether untranslated sequences in the genome of LPKV are critical determinants of its host restriction. A chimeric virus was created by inserting the entire 5′ and 3′ untranslated regions and capsid gene of LPKV into the genetic backbone of Zika virus (ZIKV). The virus replicated in mosquito cells, but not vertebrate cells. Three additional chimeras were created by exchanging specific regions in the 5′ and 3' untranslated regions of ZIKV with the corresponding regions of LPKV. A chimera that contained stem loop A (the viral promoter) of LPKV in the genetic background of ZIKV produced virus that replicated in both mosquito and vertebrate cells. These data suggest that the ZIKV NS5 polymerase recognizes the LPKV promoter and that untranslated genomic elements, other than SLA, are key determinants of insect-specific flavivirus host-specificity.

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns.

The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for more than 200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3′-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in nondepleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans-spliced, polyadenylated RNAs containing only downstream 3′-untranslated region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and DRBD18 was found in both the nucleus and the cytoplasm. We therefore suggest that in the nucleus, DRBD18 might bind to polypyrimidine tracts in 3′-UTRs of mRNA precursors. Such binding might both prevent recognition of mRNA-internal polypyrimidine tracts by splicing factors, and promote export of the processed bound mRNAs to the cytosol.

Prevalence of CAH-X Syndrome in Italian Patients with Congenital Adrenal Hyperplasia (CAH) Due to 21-Hydroxylase Deficiency.

21-hydroxylase deficiency (21OHD), the most common form of congenital adrenal hyperplasia (CAH), is associated with pathogenic variants in CYP21A2 gene. The clinical form of the disease ranges from classic or severe to non-classic (NC) or mild late onset. The CYP21A2 gene is located on the long arm of chromosome 6, within the RCCX region, one of the most complex loci in the human genome. The 3′untranslated sequence of CYP21A2 exon 10 overlap the last exon of TNXB gene (these genes lie on the opposite strands of DNA and have the opposite transcriptional direction) that encodes an extracellular matrix glycoprotein tenascin-X (TNX). A recombination event between TNXB and its pseudogene TNXA causes a 30 kb deletion producing a chimeric TNXA/TNXB gene (CAH-X chimera) where both CYP21A2 and TNXB genes are impaired. This genetic condition characterizes a subset of patients with 21OHD who display the hypermobility phenotype of Ehlers–Danlos syndrome (hEDS) (CAH-X Syndrome). The aim of this study was to assess the prevalence of CAH-X syndrome in an Italian cohort of patients with 21OHD. At this purpose, 196 probands were recruited. Multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing were used to identify the CAH-X genotype. Twenty-one individuals showed the heterozygous continuous deletion involving the CYP21A2 and part of the TNXB gene. EDS-related clinical manifestations were identified in most patients carrying the CAH-X chimera. A CAH-X prevalence of 10.7% was estimated in our population.

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design.

BackgroundAs the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive.ObjectiveTo extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently.MethodsA total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR.ResultsOut of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations.ConclusionIn addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13258-022-01264-7.

Molecular and cellular evidence for hsa-miR-1254 suppressor effect against HER2 signaling in breast cancer.

HER2 signaling upregulation is a hallmark of breast cancer which is the second cause of cancer-related death in women. Here, we were looking for the candidate microRNAs which is capable of regulating the HER2 receptor and the genes of its downstream. To this aim, preliminary bioinformatics analysis introduced hsa-miR-1254 (miR-1254) as a potential common regulator of HER2, HER3, PIK3R2, and AKT1 genes. Then, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated a lower expression level of miR-1254 in breast cancer specimens, compared to their normal pairs. Furthermore, overexpression of miR-1254 resulted in HER2, HER3, PIK3R2, and AKT1 genes downregulation, detected by RT-qPCR and confirmed by western blot analysis and enzyme-linked immunosorbent assay test against AKT1, BAX, FADD, and HER2 protein levels in SKBR3 cells. Dual-luciferase assay also supported direct interaction of miR-1254 with MREs within 3' untranslated region sequences of HER2, HER3, PIK3R2, and AKT1 target genes. Overexpression of miR-1254 in SKBR3 cells was followed by increased BAX/BCL2 expression ratio, detected by RT-qPCR, and increased proportion of G1 cell population, detected by flow cytometry. Corroborated by cell cycle analysis, MTT, Annexin V-FITC, and Live-Dead cell staining assays, overexpression of miR-1254 in MDA-MB-231 cells showed opposing results following the overexpression of miR-1254. Taken together, results indicated that miR-1254 acts as cell-type-specific tumor suppressor that targets HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR-1254 as a potential therapeutic candidate for breast cancer subtypes.

Functional assessment of miR‑1291 in colon cancer cells.

miR-1291 exerts an anti-tumor effect in a subset of human carcinomas, including pancreatic cancer. However, its role in colorectal cancer (CRC) is largely unknown. In the present study, the expression and effect of miR-1291 in CRC cells was investigated. It was identified that miR-1291 significantly suppressed the proliferation, invasion, cell mobility and colony formation of CRC cells. Additionally, miR-1291 induced cell apoptosis. A luciferase reporter assay revealed that miR-1291 directly bound the 3′-untranslated region sequence of doublecortin-like kinase 1 (DCLK1). miR-1291 also suppressed DCLK1 mRNA and protein expression in HCT116 cells that expressed DCLK1. Furthermore, miR-1291 suppressed cancer stem cell markers BMI1 and CD133, and inhibited sphere formation. The inhibitory effects on sphere formation, invasion and mobility in HCT116 cells were also explored and verified using DCLK1 siRNAs. Furthermore, miR-1291 induced CDK inhibitors p21WAF1/CIP1 and p27KIP1 in three CRC cell lines, and the overexpression of DCLK1 in HCT116 cells led to a decrease of p21WAF1/CIP1 and p27KIP1. Intravenous administration of miR-1291 loaded on the super carbonate apatite delivery system significantly inhibited tumor growth in the DLD-1 xenograft mouse model. Additionally, the resultant tumors exhibited significant upregulation of the p21WAF1/CIP1 and p27KIP1 protein with treatment of miR-1291. Taken together, the results indicated that miR-1291 served an anti-tumor effect by modulating multiple functions, including cancer stemness and cell cycle regulation. The current data suggested that miR-1291 may be a promising nucleic acid medicine against CRC.

Computational Inference, Validation, and Analysis of 5'UTR-Leader Sequences of Alleles of Immunoglobulin Heavy Chain Variable Genes.

Upstream and downstream sequences of immunoglobulin genes may affect the expression of such genes. However, these sequences are rarely studied or characterized in most studies of immunoglobulin repertoires. Inference from large, rearranged immunoglobulin transcriptome data sets offers an opportunity to define the upstream regions (5’-untranslated regions and leader sequences). We have now established a new data pre-processing procedure to eliminate artifacts caused by a 5’-RACE library generation process, reanalyzed a previously studied data set defining human immunoglobulin heavy chain genes, and identified novel upstream regions, as well as previously identified upstream regions that may have been identified in error. Upstream sequences were also identified for a set of previously uncharacterized germline gene alleles. Several novel upstream region variants were validated, for instance by their segregation to a single haplotype in heterozygotic subjects. SNPs representing several sequence variants were identified from population data. Finally, based on the outcomes of the analysis, we define a set of testable hypotheses with respect to the placement of particular alleles in complex IGHV locus haplotypes, and discuss the evolutionary relatedness of particular heavy chain variable genes based on sequences of their upstream regions.

Transcription factor CDX2 up-regulates proto-oncogenic miR-744 via a promoter activation mechanism in non-small-cell lung cancer.

BackgroundThe role of caudal-related homeobox 2 (CDX2) in the pathogenesis of non-small cell lung cancer (NSCLC) is unclear. The purpose of this study was to investigate the mRNA (message RNA) expression of CDX2 in NSCLC, and to determine its relationship with miR-744 (microRNA744) and its potential as a biomarker of NSCLC.MethodsMiR-744 is overexpressed in A549, H460, and H1299 cell lines. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression. A chromatin immunoprecipitation (ChIP) essay was performed to determine the CDX2 binding sites. We then conducted a luciferase reporter essay to analyze interaction between MiR-744 and 3'UTRs (the 3' untranslated sequences). The migration and Boyden chamber method were used to study cell mobility.ResultsIn this study, we found that ectopic CDX2 increased the expression of miR-744, while the attenuation of CDX2 reduced the expression of miR-744 by qRT-PCR. Chromatin immunoprecipitation experiments confirmed that CDX2 directly binds to the promoter of miR-744. The luciferase reporter assay further verified the binding sites of −347 to −358 bp in the most likely promoter like sequence of miR-744. CDX2-induced up-regulation of miR-744 can significantly promote the migration and invasion of NSCLC cells, while overexpression CDX2 is sufficient to rescue the migration and invasion capacity of these cells following knockdown of miR-744.ConclusionsIn summary, our results confirmed for the first time the regulatory mechanism of CDX2 on miR-744 transcription and provided a potential mechanism for CDX2 as an oncogene in lung cancer.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS – CoV-2

ABSTRACT Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3ʹ proximal genome body sequence to a 5ʹ untranslated leader sequence. This leads to a fusion between the common 5ʹ leader sequence and a 3ʹ proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.

Characterization of a New Nepovirus Infecting Grapevine.

In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California-Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological indicators. However, chlorotic ringspot symptoms were observed on sap-inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preparations from symptomatic C. quinoa revealed spherical, nonenveloped virions about 27 nm in diameter. Nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and symptomatic C. quinoa plants via high-throughput sequencing. A novel bipartite nepovirus-like genome was assembled from these contigs, and the termini of each RNA segment were verified by rapid amplification of complementary DNA ends assays. The RNA1 (7,186-nt) of the virus encodes a large polyprotein 1 of 231.1 kDa, and the RNA2 (4,460-nt) encodes a large polyprotein 2 of 148.9 kDa. Each of the polyadenylated RNA segments is flanked by 5'- (RNA1 = 156-nt; RNA2 = 170-nt) and 3'- (RNA1 = 834-nt; RNA2 = 261-nt) untranslated region sequences with >90% identities. Maximum likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences revealed the clustering of the new virus within the genus Nepovirus of the family Secoviridae. Considering its biological and molecular characteristics, and based on current taxonomic criteria, we propose that the novel virus, named grapevine nepovirus A, be assigned to the genus Nepovirus.