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Results for “noncoding RNAs”

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PubMed1991

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains.

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.

Animals↗

PubMed1986

Structural features of the 5' noncoding region of the rabbit globin messenger RNAs engaged in translation.

Accessible sites in the 5' noncoding region of the rabbit alpha- and beta-globin mRNAs were identified and compared in deproteinized RNA and in the mRNAs engaged in translation in the reticulocyte lysate. Preparations of RNA and lysate were subjected to limited nuclease digestion by RNase T1 and Neurospora endonuclease, and the cleavage sites were analyzed by a nuclease S1 mapping procedure. The free alpha-globin mRNA contained few nuclease-sensitive sites and its initiation codon AUG was masked. The free beta-globin mRNA contained a larger number of accessible sites and its AUG was highly exposed. The distribution of sensitive sites differed considerably in the lysate. In both mRNA species, a site near the 5' terminus became the one most accessible to Neurospora endonuclease. Also the accessibility of the AUG in beta-globin mRNA decreased considerably. The distribution of accessible sites in the lysate was the same when the mRNAs were undergoing rapid initiation and when initiation became limited after prolonged incubation. Inhibition of initiation by the cap analogue 7-methylguanosine 5'-triphosphate was accompanied by increased sensitivity of some of the sites in both mRNA species. One of the accessible sites in each mRNA species had a sequence complementary to the 3'-terminal portion of the 18S ribosomal RNA.

Animals↗

PubMed2026

Long noncoding RNA GAS5 disrupts intestinal epithelial barrier function by increasing small vault RNA levels.

Disruptions in the integrity of the intestinal epithelium occur commonly in inflammatory bowel disease (IBD) and critical surgical disorders, but the underlying mechanisms remain largely unknown. Here we identified long noncoding RNA GAS5 as a repressor of intestinal mucosal growth and the function of the gut epithelial barrier. The levels of tissue GAS5/Gas5 increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with IBD. Transient and tissue-specific knockdown of Gas5 in mice using CRISPR/Cas9 enhanced the renewal of the mucosa of the small intestine, increased the levels of tight junction (TJ) proteins ZO-1, ZO-2, claudin-1, and claudin-2, and improved gut barrier function. Conversely, ectopic overexpression of GAS5 in intestinal organoids and in cultured intestinal epithelium cells decreased the levels of these TJ proteins and caused epithelial barrier dysfunction. Mechanistic studies revealed that GAS5 acted as a transcriptional enhancer of the gene (2. AUTHOR: Do you mean "genes"?) encoding small noncoding vault RNAs (vtRNAs) and that GAS5 repressed TJ expression by increasing the levels of vtRNAs. Together, our results indicate that GAS5 disrupts the integrity of the intestinal epithelium by impairing mucosal growth and epithelial barrier function and that it represses TJ expression, at least in part, via vtRNAs.

Animals↗

PubMed2025

Long noncoding RNA H19 promotes the acquisition of a mesenchymal-like invasive phenotype in mesothelial primary cells through an HDAC1-mediated WT1/Sp1 switch.

Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in pro-inflammatory conditions, including peritoneal dialysis (PD), a renal replacement therapy. Characteristic of this process is the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT), a cell-specific form of EMT. Long noncoding (lnc) RNAs act as major players in physiologic regulatory circuitries of the cell. While LncRNA-H19 (lncH19), one of the first lncRNAs identified, has been broadly studied in tumorigenesis, its role in peritoneum fibrotic diseases has been scarcely addressed so far. Aim of this study was to investigate the role of H19 in the acquisition of a mesenchymal-like phenotype in primary fibrotic MCs from PD patients, and to elucidate epigenetic mechanisms controlling its expression. Genetic silencing/ectopic expression experiments revealed that H19 promoted the expression of MMT markers while downregulating the epithelial marker E-Cadherin, and favored MC directed migration and invasion on a collagen matrix. Silencing of three main H19 isoforms revealed a synergistic activity in the induction of a mesenchymal phenotype. Treatment with MS-275, an HDAC1-3 specific inhibitor previously known to promote MMT reversal, as well as HDAC1 genetic silencing, downregulated lncRNA H19 expression. Bioinformatic analysis revealed a binding sequence of Wilm's Tumor Protein 1 (WT1), the master gene of mesothelial differentiation, on the H19 promoter at an area with multiple acetylation peaks partially overlapping the binding site of Specificity protein 1 (Sp1), another transcription factor active in cellular plasticity regulation. Genetic silencing and Chromatin Immunoprecipitation (ChIP) experiments demonstrated that HDAC1 inhibition promotes a switch between WT1 and Sp1 in H19 promoter occupancy, favoring an inhibitory effect of WT1 on H19 expression and the reversal towards an epithelial-like phenotype. Overall, we discovered an HDAC1-WT1/Sp1-H19 axis potentially relevant to the design of new therapies aimed at counteracting peritoneal fibrosis.

RNA, Long Noncoding↗

PubMed1991

Structure and expression of mouse germline immunoglobulin gamma 3 heavy chain transcripts induced by the mitogen lipopolysaccharide.

Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

Animals↗

PubMed2025

Association between residential greenness and coronary heart disease: A proteomics and miRNA microarray analysis.

Greenness has been linked to cardiovascular disease. However, the specific biological mechanisms through which greenness impacts coronary heart disease (CHD) remain unclear. We aim to explore the underlying epigenetic mechanisms linking greenness and CHD by using proteomics and miRNA microarray. A total of 2387 participants were included in the population study, 816 of whom were diagnosed with CHD. Residential greenness exposure was characterized using the normalized difference vegetation index (NDVI). Generalized additive models and restricted cubic splines investigated the association between greenness and CHD. Mediation analysis examined whether cardiovascular metabolic risk factors (blood pressure, inflammation indicators, and glucose) mediated the association. After proteomics and miRNA microarray screening, Elisa and qRT-PCR validated selected proteins (THBS1, FCN3, and LTBP1) and miRNAs (miR-671-5p, miR-124-3p, and miR-379-5p) in CHD. Among these, LTBP1 and miR-379-5p showed significant differential expression (P < 0.05) and were examined as potential molecular mediators. Higher greenness exposure within a 1000-m area was associated with a lower risk of CHD (OR: 0.86, 95 % CI: 0.81, 0.92). Systolic blood pressure (6.32 % [95 % CI: 1.49 %, 13.12 %]), lymphocyte (10.98 % [95 % CI: 3.76 %, 22.00 %]), monocyte (9.94 % [95 % CI: 3.42 %, 20.87 %]), and fasting blood glucose (3.41 % [95 % CI: 0.56 %, 7.84 %]) mediated this association. LTBP1 and miR-379-5p were differentially expressed in CHD and mediated 7.19 % [95 % CI: 0.01 %, 23.37 %] and 20.03 % [95 % CI: 2.85 %, 69.71 %] of greenness effect on CHD, respectively. Combining the population study and experiments, we found that miR-379-5p and LTBP1 may jointly modulate vascular constriction and immune inflammation in the association between greenness and CHD.

Humans↗

PubMed2024

Stroke genetics and how it Informs novel drug discovery.

INTRODUCTION: Stroke is one of the main causes of death and disability worldwide. Nevertheless, despite the global burden of this disease, our understanding is limited and there is still a lack of highly efficient etiopathology-based treatment. It is partly due to the complexity and heterogenicity of the disease. It is estimated that around one-third of ischemic stroke is heritable, emphasizing the importance of genetic factors identification and targeting for therapeutic purposes. AREAS COVERED: In this review, the authors provide an overview of the current knowledge of stroke genetics and its value in diagnostics, personalized treatment, and prognostication. EXPERT OPINION: As the scale of genetic testing increases and the cost decreases, integration of genetic data into clinical practice is inevitable, enabling assessing individual risk, providing personalized prognostic models and identifying new therapeutic targets and biomarkers. Although expanding stroke genetics data provides different diagnostics and treatment perspectives, there are some limitations and challenges to face. One of them is the threat of health disparities as non-European populations are underrepresented in genetic datasets. Finally, a deeper understanding of underlying mechanisms of potential targets is still lacking, delaying the application of novel therapies into routine clinical practice.

Humans↗

PubMed1991

Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.

We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.

Base Sequence↗

PubMed1990

Factors affecting efficient infection of tobacco with in vitro RNA transcripts from cloned cDNAs of satellite tobacco mosaic virus.

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindIII or Pstl restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3' noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.

Base Sequence↗

PubMed2026

Mapping the regulatory architecture of circadian clock adaptation: A genome-wide eQTL analysis in Drosophila melanogaster.

The circadian clock enables organisms to align internal daily rhythms with environmental cues, with major consequences for survival and fitness. Although the molecular framework of this system in Drosophila melanogaster is well characterized through transcription translation feedback loops involving ten core clock genes, the genetic basis of natural variation in their expression remains poorly understood. Here, we used natural expression variation to identify expression quantitative trait loci (eQTLs) through genome-wide association mapping. Using the Drosophila Genetic Reference Panel, we measured relative expression of all core clock genes at a single time point two hours after light onset. We identified 109 significant SNPs and 28 indels associated with expression variation across the clock network. Expression levels varied widely, with Pdp1ε showing the greatest variation (an 86-fold difference between extreme lines) and cyc the least (11.3-fold). Only three significant SNPs were located within clock genes themselves, all in Clk, whereas most associations represented trans-eQTLs in genes with diverse molecular functions. Candidate regulators included transcription factors such as Abd-B, tai, and E5; RNA binding proteins including Pum, Bru-3, and Mbl; and several long noncoding and antisense RNAs. Variants were also detected in gbb and the BMP pathway transcription factor Mad. Consistent with this, Mad knockdown reduced vri expression. Together, these results reveal a complex regulatory architecture underlying natural variation in circadian gene expression.

Journal Article↗

PubMed1992

Mutagenesis of a hexanucleotide sequence conserved in potexvirus RNAs.

Biologically active in vitro transcripts were synthesized from a cloned cDNA of a defective RNA (D RNA) of clover yellow mosaic virus (CYMV) and were used to determine if a hexanucleotide motif (5'-ACUUAA) conserved in the 3' noncoding region of potexvirus RNAs is essential for accumulation of progeny D RNA in planta. Deletion of rearrangement of the entire hexanucleotide sequence in the D RNA resulted in no detectable accumulation of progeny D RNA when coinoculated with helper CYMV RNA. Single-base substitutions of the four central nucleotides of the hexanucleotide sequence revealed that viable D RNAs can tolerate single residue changes at positions 3 and 5 only. These results suggest that the hexamer motif is involved in the process of D RNA propagation. The hexanucleotide sequence was also identified in the negative strand of potexvirus RNAs in the regions proposed to represent subgenomic RNA (sgRNA) promoters. In addition, the hexamer motif is present in similar regions in carlavirus RNAs. The conservation of this hexanucleotide (in orientation and position) in potexvirus and carlavirus RNAs, which serve as templates for full-length negative-strand synthesis and sgRNA production, strongly suggests that it plays a functional role in the synthesis of viral RNAs. Taken together, our data support our previous proposal (Bancroft et al., 1991. J. Gen. Virol. 72, 2173-2181) that the hexanucleotide sequence acts as a cis element involved in synthesis of full-length negative-sense viral RNA and further implicate the sequence in a similar role in production of positive-sense sgRNA.

Capsid↗

PubMed1991

Biological activities of hybrid RNAs generated by 3'-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.

Cloning, Molecular↗

PubMed1991

Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5' noncoding region.

Hepatitis A virus (HAV) RNAs were translated in vitro in rabbit reticulocyte lysates. The pattern of proteins synthesized from full-length HAV RNA was highly complex, consisting of a continuous spectrum of polypeptides ranging from less than 20,000 to greater than 200,000 Da. The pattern was not significantly altered by varying incubation times, ion, or other reaction parameters, or by the addition of HeLa or BS-C-1 cell extracts to the translation reactions. Plasmids engineered with mutations in the 3C coding region produced transcripts which directed the synthesis of the same overall pattern of polypeptide products as those transcribed from wild-type sequences, suggesting that protein processing by 3C did not generate the complex set of protein products. Translation of RNA containing only the P3 coding region of HAV, directly adjacent to the HAV 5' noncoding region, generated a set of protein products which precisely matched a subset of those synthesized from full-length HAV RNA. The translation products of P3 RNA, full-length RNA, and mutant 3C-containing RNAs were analyzed by immunoprecipitation with antisera specific for 3D, VP1, and 2C sequences; several products were subjected to N-terminal sequence analysis. All together, the results demonstrate that translation of HAV RNA in rabbit reticulocyte lysates initiates predominantly at a large number of internal AUG codons, especially those in the P3 coding region. A minor population of products is initiated from sites in the P1 and P2 regions. The latter proteins undergo some proteolytic processing, at unidentified sites, catalyzed by 3C protein sequences. Replacement of the HAV 5' noncoding region with encephalomyocarditis virus 5' end sequences increased initiation at the correct polyprotein start site and both reduced and altered the products generated by internal initiation.

3C Viral Proteases↗

PubMed1988

Insights into Theiler's virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains.

Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which can be divided into two subgroups based primarily on their neurovirulence after intracerebral inoculation: the highly virulent GDVII group and the less virulent TO strains. To begin to elucidate the molecular basis of neurovirulence of the two TMEV subgroups, we have cloned and sequenced the entire 8105 nucleotide RNA genome of the highly virulent GDVII virus and compared it to the less virulent BeAn 8386 virus (D. C. Pevear, M. Calenoff, E. Rozhon, and H. L. Lipton (1987) J. Virol. 61, 1507-1516). The viruses are 90.4% identical at the nucleotide level. The highest level of nucleotide identity is in the 5' and 3' noncoding regions of the RNAs (95.5 and 99.2%, respectively): regions believed to be important for control of viral RNA synthesis, initiation of translation, encapsidation, and virion uncoating. The 2303 amino acid polyproteins of BeAn and GDVII viruses are 95.7% identical at the amino acid level (99 of 2303 residues differed). Thirty-nine of these amino acid differences occur in the three surface coat proteins, VP1 (20 differences), VP2 (10 differences), and VP3 (9 differences), while the remainder of the changes are distributed throughout the polyprotein. Although these levels of identity are too low to determine where neurovirulence maps based solely on nucleotide sequence analysis, having the complete sequence will facilitate construction of recombinant BeAn-GDVII viruses to be used for this purpose.

Amino Acid Sequence↗

PubMed1990

Translational regulation of the immunoglobulin heavy-chain binding protein mRNA.

Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picornaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mechanism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.

Carrier Proteins↗

PubMed1979

Nucleotide sequence of the 3'-noncoding region of alfalfa mosaic virus RNA 4 and its homology with the genomic RNAs.

A 226-nucleotide fragment was derived from alfalfa mosaic virus RNA 4 (ALMV RNA 4), the subgenomic messenger for viral coat protein, and its sequence was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. The fragment contains the 3'-terminal 45 nucleotides of the coat protein cistron and the complete 3'-noncoding region of 182 nucleotides. The total length of RNA 4 was calculated to be 881 nucleotides. AlMV RNAs 1, 2 and 3 were elongated with a 3'-terminal poly(A) stretch and subjected to sequence analysis by using a specific primer, reverse transcriptase and chain terminators. This revealed and extensive homology between the 3'-terminal 140 to 150 nucleotides of all four ALMV RNAs. Despite a number of base substitutions, the secondary structure of the homologous region is highly conserved. The observed homology indicates that, as with RNA 4, the sites with a high affinity for the viral coat protein are located at the 3'-termini of the genomic RNAs.

Base Sequence↗

PubMed1988

Sequence and secondary structure analysis of the 5'-terminal region of flavivirus genome RNA.

The 5'-terminal noncoding region sequences were determined for the genome RNAs of seven strains of St. Louis encephalitis virus (SLEV) and one strain of West Nile virus (WNV) using a single synthetic cDNA primer complementary to the 5'-terminus of the coding region of a strain of WNV RNA. The 5'-terminal sequences obtained for the SLEV and WNV RNAs were compared with published sequences for yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), and dengue virus. While only short regions within the 5'-noncoding sequence were conserved among different flavivirus RNAs, significant homology was observed in this region among members of the same flavivirus subgroup and almost complete conservation was observed between different strains of the same virus. For example, seven strains of SLE, isolated from different geographic locations over a 17-year period and differing in their neurovirulence phenotype, contained only two to four nucleotide changes in the 5'-noncoding region. Interestingly, each of three low-virulence strains shared the same unique base substitution at position 16. Secondary structures predicted to be formed by the 5'-termini of each of the different flavivirus genome RNAs were of similar size and shape, in each case consisting of a stem with a small top loop and a larger side loop. The prediction of a common structure among a number of different flaviviruses, despite the lack of extensive sequence homology, suggests that this secondary structure is functionally important. An additional stem and loop structure is predicted to be formed in the region spanning the translation initiation codon. This structure showed significantly less conservation of size and shape than the 5'-terminal secondary structure.

Base Sequence↗

PubMed1990

Pseudoknots: a new motif in the RNA game.

In the last few years a novel RNA folding principle called pseudoknotting has emerged. Originally discovered in noncoding regions of plant viral RNAs, pseudoknots now appear to be a widespread structural motif in a number of functionally different RNAs. These structural elements are part of tRNA-like structures and are involved in folding catalytic sites of ribozymes. They increase the efficiency of ribosomal frameshifting or can serve as specific binding sites for regulatory proteins.

Base Sequence↗