UTR Sequences Research Articles

Reconstitution and analyses of RNA interactions with eukaryotic translation initiation factors and ribosomal preinitiation complexes.

Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5' UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of the effects of labeled and unlabeled mRNA recruitment factors on both the rate and factor dependence of mRNA recruitment to the translation preinitiation complex in response to RNA sequence- and structure-changes. In addition to providing streamlined and detailed protocols, we describe a new construct for purification of higher yields of fluorescently labeled and unlabeled full-length eIF4G.

Occurrence of Salivirus in Sewage and River Water Samples in Karaj, Iran.

Salivirus is a newly discovered virus which seems to be related to acute gastroenteritis in children. Salivirus may infect susceptible children by fecal-oral route after exposure to contaminated water. The present study aims to evaluate the occurrence and quantity of Salivirus in treated and untreated sewage water and river water samples collected in the city of Karaj, Iran by reverse transcription-quantitative PCR assay. A total of 50 samples were collected from environmental waters containing 22 treated and untreated sewage water in volume of 1l and 28 river water samples in volume of 5l were included in this study. After viral RNA extraction, the Real-time PCR was performed to amplify the 5'UTR sequence of Salivirus genome and viral load was assessed. Out of the 50 samples tested, the Salivirus genomic RNA was identified in 5/12 (41.6%) of treated and 3/10 (30%) of untreated sewage samples and in 8/28 (28.5%) of river water samples. The maximum viral load was 4.8 × 106 copies/l in treated sewage water sample in September and the lower viral load was 4 × 105 copies/l related to treated sewage water taken in December. This is the first report of Salivirus occurrence in the environmental waters in Iran. The viral prevalence of Salivirus in each of the three sets of tested samples was within low to moderate in range.

MiR-296-5p suppresses papillary thyroid carcinoma cell growth via targeting PLK1.

Our study aimed to explore the effects of miRNA-296-5p on the biological behaviors of papillary thyroid carcinoma (PTC) cells and its potential mechanism. Twenty-eight PTC tissues and the corresponding non-cancerous tissues were collected. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) analysis was performed to detect the expression levels of miR-296-5p in PTC tissues and the adjacent non-cancerous tissues. Besides, the different endogenous expression levels of miR-296-5p in PTC cell line (K1) and normal thyroid gland cell line (Nthy-ori3-1) were also detected by RT-qPCR. Bioinformatics analysis, Western blot and Dual-Luciferase reporter gene assay were performed to demonstrate whether polo-like kinase 1 (PLK-1) was a downstream target of miR-296-5p. Subsequently, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, flow cytometry analysis, colony formation assay and TUNEL assay were performed to estimate whether PLK1 down-regulation could attenuate the malignant behaviors of PTC cells in vitro. RT-qPCR results showed that the expression level of miR-296-5p was significantly down-regulated in PTC tissues and cells, indicating that miR-296-5p may participate in PTC development. We predicted target genes of miR-296-5p by bioinformatics and identified PLK1 as a target gene of miR-296-5p. By Western blot and Dual-Luciferase reporter gene assay, we confirmed that miR-296-5p was partially complement to PLKl mRNA 3'UTR sequence and inhibited PLK1 expression at the post-transcriptional level. In vitro experiments suggested that the transfection of miR-296-5p mimics into K1 cells suppressed cell proliferation, inhibited cell clone formation, arrest the cell cycle in G2/M phase and induced apoptosis. Importantly, PLK1 reversed the inhibitory effects of miR-296-5p on biological behaviors of PTC. MiR-296-5p influences the biological behaviors of PTC by regulating PLK1. These findings provide a new perspective for the molecular mechanism of PTC pathogenesis and also contribute to developing new targets and methods for PTC treatment.

MicroRNA-26a-2 maintains stress resiliency and antidepressant efficacy by targeting the serotonergic autoreceptor HTR1A

The association between dysregulated serotonergic activity and major depressive disorder (MDD) is well known. However, the various mechanisms underlying serotonergic dysregulation in MDD remain unclear. Previous research on serotonergic (5-HT) neurons identified microRNA-26a (miR-26a) targeting of the serotonin autoreceptor, 5-HT receptor 1A (HTR1A). Reporter assays with the Htr1a 5′UTR sequence were performed in vitro. Adult transgenic mouse models altering miR-26a-2 and Htr1a expression were used for chronic social defeat, antidepressant treatment, and in vivo lentiviral experiments. Mice were tested for anxiety-like behavior using the elevated plus-maze, dark-light transfer, and open-field tests, and for depression-like behavior using the forced-swim test. We confirmed that miR-26a-2 downregulates Htr1a expression in 5-HT neurons in vitro. miR-26a-2 levels were significantly upregulated in the mouse dorsal raphe nucleus (DRN) following antidepressant therapy. The transgenic murine model overexpressing miR-26a-2 in serotonergic neurons displayed improved behavioral resiliency to social defeat. These effects were abrogated by the addition of Htr1a overexpression. In contrast, the transgenic murine model with miR-26a-2 knockdown in serotonergic neurons displayed increased anxious behavior and weakened antidepressant response. These effects were rescued by silencing Htr1a expression. Our findings suggest that miR-26a-2 functions as an endogenous antidepressant by targeting HTR1A in serotonergic neurons.

HLA-A promoter, coding, and 3'UTR sequences in a Brazilian cohort, and their evolutionary aspects.

HLA-A is the second most polymorphic locus of the human leucocyte antigen (HLA) complex encoding a key molecule for antigen presentation and NK cell modulation. Many studies have evaluated HLA-A variability in worldwide populations, focusing mainly on exons, but the regulatory segments have been poorly characterized. HLA-A variability is particularly high in the segment encoding the peptide-binding groove (exons 2 and 3), which is related to the antigen presentation function and the balancing selection in these segments. Here we evaluate the genetic diversity of the HLA-A gene considering a continuous segment encompassing the extended promoter (1.5 kb upstream of the first translated ATG), all exons and introns, and the entire 3' untranslated region, by using massively parallel sequencing. To achieve this goal, we used a freely available bioinformatics workflow that optimizes read mapping for HLA genes and defines complete sequences using either the phase among variable sites directly observed in sequencing data and probabilistic models. The HLA-A variability detected in a highly admixed population sample from Brazil shows that the HLA-A regulatory segments present few, but divergent sequences. The regulatory segments are in close association with the coding alleles. Both exons and introns are highly variable. Moreover, patterns of molecular diversity suggest that the promoter, in addition to the coding region, might be under the same selective pressure, but a different scenario arises when it comes to exon 4 and the 3'UTR segment.

Regulat-INGs in tumors and diseases: Focus on ncRNAs

ING family genes (Inhibitor of Growth) are tumor suppressor genes that play a vital role in cell homeostasis. It has been shown that their expression is lost or diminished in many cancers and other diseases. The main mechanisms by which they are regulated in oncogenesis have not yet been fully elucidated. The involvement of non-coding RNAs (ncRNAs) and in particular microRNAs (miRNAs) in post-transcriptional gene regulation is well established. miRNAs are short sequences (18–25 nucleotides) that can bind to the 3 ′UTR sequence of the targeted messenger RNA (mRNA), leading to its degradation or translational repression. Interactions between the ING family and miRNAs have been described in some cancers but also in other diseases. The involvement of miRNAs in ING family regulation opens up new fields of investigation, particularly for targeted therapies. In this review, we will summarize the regulatory mechanisms at the RNA and protein level of the ING family and focus on the interactions with ncRNAs.

Synchronous Detection of BPV and BVDV with Duplex Taqman qPCR Method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrhea in dairy herds. BPV is a member of bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirus genus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes according to the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viral diarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the present study was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV. Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved 5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed in vitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, and then used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probe qRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were 2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplification conditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTR gene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. For clinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealing temperature was achieved in 43.2 ℃ fro duplex BPV and BPIV. Dynamic curves and standard curves were created following amplification of recombinant plasmids using the optimized duplex Taqman BPV and BVDV, with an amplification efficiency of 95.69%. Duplex Taqman qPCR could only detect DNA of BPV and cDNA of BVDV with a strong specificity. The detection limitation was as low as 2.0 × 102 copies/μL of pMD18-T-BPV plasmid and 2.0 × 101 copies/μL for pMD18-T-BVDV plasmid, respectively. Sensitivity of detection was 100-fold higher than conventional PCR. Duplex Taqman qPCR had excellent repeatability or stability with less than 1.2% of intra-assay and inter-assay. 35 and 47 positive feces samples were identified using duplex Taqman qPCR in comparison to 30 and 42 positives for universal PCR, respectively. Discussion: The bovine viral diarrhea virus (BVDV) is a key pathogenic factor in bovine diarrhea. Currently, few effective measures are available for the treatment or prevention for BVDV and BPV infections in animals. The technique was proven to be repeatable and linear over a range of at least 5 magnitudes, from 101 to 105 RNA/DNA copies, thus ensuring an accurate measurement of BPV DNA and BVDV RNA loads in clinical samples. In conclusion, a duplex Taqman qPCR was established for detecting simultaneously BPV and BVDV. Taqman qPCR method was rapid and specific assay. This assay was 100-fold sensitive than conventional PCR. It will be propitious to rapidly and differentially diagnose pathogens of viral diarrhea of dairy farms. Taqman qPCR method was rapid and specific assay and had a sensitivity of 2.0 copies/μL.

Hsa-miR-5195-3P induces downregulation of TGFβR1, TGFβR2, SMAD3 and SMAD4 supporting its tumor suppressive activity in HCT116 cells.

MicroRNAs are classified as small non-coding RNAs that regulate gene expression mainly through targeting the 3'UTR region of mRNAs. A great number of miRNAs play important role in the regulation of signaling pathways in normal and cancer cells. Here, we predicted hsa-miR-5195-3p (miR-5195-3p) as a potential regulator of TGFβ signaling and investigated its effect on TGFB-R1, TGFB-R2, SMAD2, SMAD3 and SMAD4 transcripts which are key players of TGFβ/SMAD signaling pathway. Overexpression of miR-5195 in HCT116 cells resulted in a significant reduction of TGFB-R1, SMAD2, SMAD3, and SMAD4 at the mRNA level which was confirmed using RT-qPCR. Consistently, western blot analysis confirmed that miR-5195 overexpression in HCT116 cells resulted in downregulation of TGFBR1 at the protein level. Furthermore, dual luciferase analysis verified the direct interaction of miR-5195 with TGFB-R1 and SMAD4 3'UTR sequences in SW480 cells. Additionally, flow cytometry analysis confirmed that miR-5195 overexpression significantly increased the sub-G1 and decreased the G-1 cell populations in both SW480 and HCT116 cell lines. Finally, miR-5195 overexpression significantly downregulated c-MYC and cyclin D1 but upregulated p21 genes. Overall, our results indicated that miR-5195 modulates TGFβ signaling pathway and affects the cell cycle progression through targeting TGFB-R1, TGFB-R2, SMAD2, SMAD3, SMAD4 transcripts.

MicroRNA-20b promotes proliferation of H22 hepatocellular carcinoma cells by targeting PTEN.

MicroRNAs (miRNAs/miRs) are small, noncoding RNA molecules that are closely associated with the occurrence and development of tumors. miR-20b is overexpressed in hepatocellular carcinoma cell lines and tissues. However, it is not clear whether miR-20b can promote the proliferation of hepatocellular carcinoma cells. In the present study, the proliferation of H22 mouse hepatocellular carcinoma cells was detected using the Cell Counting Kit-8 assay. MiRanda software was used to predict the binding sites of miR-20b to the 3'-untranslated region (3'-UTR) of phosphatase and tensin homolog (PTEN). The 3'-UTR sequence of the PTEN gene was amplified using the polymerase chain reaction in H22 cells. The recombinant plasmid or empty plasmid was co-transfected with miR-20b mimics or miR-20b scramble into HeLa cells, and luciferase activity was assessed by Dual-Luciferase® Reporter Assay System 24 h post-transfection. In the present study, miR-20b knockdown significantly inhibited the proliferation of H22 mouse hepatocellular carcinoma cells. In addition, miR-20b inhibition upregulated the expression of PTEN, and it was revealed that miR-20b may directly target the 3'-untranslated region of the PTEN gene. Downregulation of PTEN partially reversed the anti-proliferative effect of miR-20b on H22 cells. In conclusion, miR-20b may promote H22 cell proliferation by targeting PTEN, providing a rationale for further study investigating novel therapeutic strategies for liver cancer.

Incidence and genetic diversity of apple chlorotic leaf spot virus in Iran

Apple chlorotic leaf spot virus (ACLSV) is one of the most common viruses infecting apple and pear trees, and Iran is among the top ten apple-producing countries in the world. In the present study, incidence and genetic diversity of ACLSV were investigated in the main apple-growing regions of Iran. To achieve this purpose, a total of 1053 leaf samples were collected from orchards located in nine Iranian provinces. ACLSV infection was detected by DAS-ELISA in 48 samples (4.55%) from seven provinces and was confirmed by RT-PCR. Based on the geographical origin, 19 representative isolates were selected for phylogenetic analysis. Nineteen amplicons, with a size of 677 base pairs (bp), containing the 3′ end of the movement protein (MP), the coat protein (CP) gene, and 3′-UTR sequence, were sequenced. Sequence analysis, using data of 45 isolates from 12 different countries, including the 19 Iranian isolates, showed that CP gene among the Iranian isolates were 80–99% identical at both nucleotide and amino acid levels, and these isolates were placed in the B6 (AB326224) and P-205 (D14996) ACLSV groups. This is the first genetic analysis of ACLSV in Iran.

Outbreak of myelocytomatosis caused by mutational avian leukosis virus subgroup J in China, 2018.

Avian leukosis virus subgroup J (ALV-J) was isolated in meat-type breeder chickens for the first time in 1988 in the United Kingdom. Due to the application of an eradication program, there were fewer reports related to myelocytomatosis or ALV-J in China after 2013. However, there was another breakout almost simultaneously in six provinces of China in February 2018. On-site, 15- to 20-week-old broiler breeder chickens showed depression, paralysis and weight loss. Mortality for certain flocks reached 15%. Sick chickens showed numerous yellow-white neoplasms growing in the sternum, rib and lumbar vertebra and had hepatic and renal metastasis. Histopathological observation showed all neoplasms were myelocytomas, and there were massive myelocyte-like tumour cells in the liver, kidney and bone marrow. To explore the aetiology of this re-outbreak of myelocytomatosis in China, we collected tumour-bearing chickens and isolated six strains of ALV-J (GM0209-1 to -6). Phylogenetic analysis of gp85 and gp37 showed GM0209 strains were clearly distinct from the prototype strain of ADOL-7501, HPRS-103 and NX0101, and there was a mutation, R176G, in the conserved region between hr1 and hr2 regions of gp85, which was not found in other 44 ALV-J strains. The 3'UTR nucleotide sequences of GM0209 isolates showed there was a signature deletion of 11 nt that was also present in 3'UTR sequences of SCDY1 and NHH, two isolates that have a reported association with haemangioma, indicating this deletion could not determine the tumour type induced by ALV-J. Although the eradication program of ALV-J has been successfully applied in China, the outbreak of ALV-J still occurred, and the virus strain spread quickly. Thus, the biocharacteristics and pathogenesis of mutational ALV-J should be further studied.

Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5'UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication.

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication.

Systematic Evolution of Ligands by Exosome Enrichment: A Proof-of-Concept Study for Exosome-Based Targeting Peptide Screening.

Selection of a peptide that binds preferentially to targeted cells or tissues is a prerequisite for targeted therapy. Although in vivo phage display is a high-throughput method, it is restricted in identifying target ligands specific for different vascular beds. In this study, the exosomes are repurposed for targeting peptide screening. Briefly, the signal peptide region of Lamp2b (a membrane protein on the exosomes) in the N-terminus is engineered to fuse with 10 aa long random peptides, while the C-terminus of Lamp2b is fused with the MS2 coating protein (MCP). Then, the whole Lamp2b-MCP open reading frame (ORF) is further engineered to harbor a 3'UTR sequence consisting of MS2. The resultant exosomes from engineered Lamp2b-MCP expressing cells display the 10 aa peptides on the outside while containing the genetic information inside. By proof-of-principle experiments, the exosomes with different peptides could preferentially distribute to different tissues besides the spleen and liver. Furthermore, detailed target sequences for different tissues are enriched by rounds of selection. In summary, the established novel targeted peptide screening strategy, namely, "exosome display," has broad applicability, especially for displaying and screening targeted peptides for the cells outside the capillary with condense barriers, like the neurons in the brain.

Functional validation of microRNA-126-3p as a platelet reactivity regulator using human hematopoietic stem cells

Platelets are an abundant source of microRNAs (miRNAs), which may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, are pointed out as potential biomarkers of platelet reactivity and recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain and functional validation of these candidate miRNAs on human-derived cells is lacking. To functionally validate miR-126-3p as regulator of platelet reactivity in platelet-like structures derived from human hematopoietic stem cells. The functional impact of miRNAs on platelet-like structures derived from human hematopoietic stem cells was evaluated using a flow-based assay. CD34+ derived megakaryocytes were transfected with selected miRNA or siRNA and were differentiated in platelet-like structures. Platelet adhesion phenotype was assessed by perfusion of differentiated cells in a microfluidic system under a constant shear rate. Overexpression of miR-126-3p increased platelet-like structures adhesion compared to control. Moreover, miR-126-3p transfection was associated with downregulation of ADAM9, a validated target of miR-126-3p, and of PLXNB2, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results than miR-126-3p transfection. Reporter gene assay confirm that miR-126-3p directly targets PLXNB2 3′UTR sequence. Taken together, using a flow-based assay, we functionally validate miR-126-3p as a regulator of platelet reactivity on platelet-like structures derived from human hematopoietic stem cells. Moreover, PLXNB2 was identified as a platelet reactivity regulator supporting the hypotheses that miR-126-3p modulates platelet reactivity at least in part by downregulation of PLXNB2.

Regulatory effect of hsa-miR-5590-3P on TGFβ signaling through targeting of TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 transcripts

Transforming growth factor-β (TGFβ) signaling acts as suppressor and inducer of tumor progression during the early and late stages of cancer, respectively. Some miRNAs have shown a regulatory effect on TGFβ signaling and here, we have used a combination of bioinformatics and experimental tools to show that hsa-miR-5590-3p is a regulator of multiple genes expression in the TGFβ signaling pathway. Consistent with the bioinformatics predictions, hsa-miR-5590-3p had a negative correlation of expression with TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 genes, detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the dual luciferase assay supported the direct interaction between hsa-miR-5590-3p and TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4-3'UTR sequences. Consistently, the TGFβ-R1 protein level was reduced following the overexpression of hsa-miR-5590-3p, detected by Western analysis. Also, hsa-miR-5590-3p overexpression brought about the downregulation of TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 expression in HCT-116 cells, detected by RT-qPCR, followed by cell cycle arrest in the sub-G1 phase, detected by flow cytometry. RT-qPCR results indicated that hsa-miR-5590-3p is significantly downregulated in breast tumor tissues (late stage) compared to their normal pairs. Altogether, data introduces hsa-miR-5590-3p as a negative regulator of the TGFβ/SMAD signaling pathway which acts through downregulation of TGFβ-R1, TGFβ-R2, SMAD3 and SMAD4 transcripts. Therefore, it can be tested as a therapy target in cancers in which the TGFβ/SMAD pathway is deregulated.

MicroRNAs as Regulators of Insulin Signaling: Research Updates and Potential Therapeutic Perspectives in Type 2 Diabetes.

The insulin signaling pathway is composed of a large number of molecules that positively or negatively modulate insulin specific signal transduction following its binding to the cognate receptor. Given the importance of the final effects of insulin signal transduction, it is conceivable that many regulators are needed in order to tightly control the metabolic or proliferative functional outputs. MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively modulate gene expression through their specific binding within the 3′UTR sequence of messenger RNA (mRNA), thus causing mRNA decoy or translational inhibition. In the last decade, miRNAs have been addressed as pivotal cellular rheostats which control many fundamental signaling pathways, including insulin signal transduction. Several studies demonstrated that multiple alterations of miRNAs expression or function are relevant for the development of insulin resistance in type 2 diabetes (T2D); such alterations have been highlighted in multiple insulin target organs including liver, muscles, and adipose tissue. Indirectly, miRNAs have been identified as modulators of inflammation-derived insulin resistance, by controlling/tuning the activity of innate immune cells in insulin target tissues. Here, we review main findings on miRNA functions as modulators of insulin signaling in physiologic- or in T2D insulin resistance- status. Additionally, we report the latest hypotheses of prospective therapies involving miRNAs as potential targets for future drugs in T2D.

Cell Surface Antigen Display for Neuronal Differentiation-Specific Tracking.

Cell therapeutic agents for treating degenerative brain diseases using neural stem cells are actively being developed. However, few systems have been developed to monitor in real time whether the transplanted neural stem cells are actually differentiated into neurons. Therefore, it is necessary to develop a technology capable of specifically monitoring neuronal differentiation in vivo. In this study, we established a system that expresses cell membrane-targeting red fluorescent protein under control of the Synapsin promoter in order to specifically monitor differentiation from neural stem cells into neurons. In order to overcome the weak expression level of the tissue-specific promoter system, the partial 5′ UTR sequence of Creb was added for efficient expression of the cell surface-specific antigen. This system was able to track functional neuronal differentiation of neural stem cells transplanted in vivo, which will help improve stem cell therapies.

Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5'-UTRs.

The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.

LncRNA NEAT1 facilitates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs) via targeting miR-377 and upregulating SIRT1, VEGFA, and BCL-XL

ObjectiveThe present study was designed to investigate the mechanism by which lncRNA NEAT1 regulates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). MethodsOGD-treated BMECs were used to mimic cerebral ischaemia in vitro. The expression of lncRNA NEAT1 and miR-377 and proteins including VEGFA, SIRT1, and BCL-XL were measured by real-time quantitative PCR (qRT-PCR) and western blot, respectively. Cell viability and caspase 3 activity of BMECs under different conditions were determined using MTT and caspase activity assays, respectively. Matrigel-based angiogenesis assays were employed to evaluate the effect of lncRNA NEAT1 on angiogenesis. A dual-luciferase reporter assay was used to validate direct binding of miR-377 to putative targets. ResultsOGD exposure reduced the cell viability of BMECs. Upregulation of lncRNA NEAT1 and downregulation of miR-377 were also observed under OGD conditions. Knockdown of lncRNA NEAT1 inhibited angiogenesis and aggravated apoptosis in OGD-induced BMECs. Meanwhile, the expression level of miR-377 was upregulated while its downstream targets (VEGFA, SIRT1, and BCL-XL) were downregulated after lncRNA NEAT1 knockdown. Furthermore, miR-377 inhibited the angiogenesis and survival of OGD-induced BMECs. The expression of VEGFA, SIRT1, and BCL-XL were all attenuated by miR-377 overexpression. The dual-luciferase reporter assay proved miR-377 targeted the 3′ UTR sequences of lncRNA NEAT1, VEGFA, SIRT1, and BCL-XL. ConclusionlncRNA NEAT1 facilitated the survival and angiogenesis of OGD-induced BMECs via targeting miR-377 and promoting the expression of VEGFA, SIRT1, and BCL-XL, suggesting that lncRNA NEAT1 could be a promising target for cerebral ischaemia treatment.

The role of RNA structure in translational regulation by L7Ae protein in archaea.

A recent study has shown that archaeal L7Ae binds to a putative k-turn structure in the 5′-leader of the mRNA of its structural gene to regulate translation. To function as a regulator, the RNA should be unstructured in the absence of protein, but it should adopt a k-turn-containing stem–loop on binding L7Ae. Sequence analysis of UTR sequences indicates that their k-turn elements will be unable to fold in the absence of L7Ae, and we have demonstrated this experimentally in solution using FRET for the Archaeoglobus fulgidus sequence. We have solved the X-ray crystal structure of the complex of the A. fulgidus RNA bound to its cognate L7Ae protein. The RNA adopts a standard k-turn conformation that is specifically recognized by the L7Ae protein, so stabilizing the stem–loop. In-line probing of the natural-sequence UTR shows that the RNA is unstructured in the absence of L7Ae binding, but folds on binding the protein such that the ribosome binding site is occluded. Thus, L7Ae regulates its own translation by switching the conformation of the RNA to alter accessibility.