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PubMed1983

Cloning of mouse carbonic anhydrase mRNA and its induction in mouse erythroleukemic cells.

Mouse carbonic anhydrase mRNA was detected in poly(A+) RNA of anemic spleens sedimenting as a RNA species at 14 S. Subsequently, poly(A+) RNA (12-16 S) was used as a template for the synthesis of double-stranded cDNA, which was inserted into the PstI site of pBR322 by oligo-dG:dC tailing. A recombinant plasmid containing carbonic anhydrase cDNA was identified by a positive hybridization selection assay and by partial DNA sequencing. Predicted amino acid sequences showed homology with the known sequences of rabbit and human carbonic anhydrase I and II. The clone contained sequences for most of the coding region and 600-700 base pairs at the 3' noncoding region of the mRNA. Hybridization analysis of poly(A+) RNA from uninduced and induced mouse erythroleukemic cells labeled for short and long time periods indicated that induction results in an increase of carbonic anhydrase mRNA in newly synthesized RNA.

Animals↗
PubMed2026

Nuclear exosome targeting complexes modulate cohesin binding and enhancer-promoter interactions in 3D.

Three-dimensional long-range contacts between enhancers and promoters are thought to be largely determined by loop extrusion driven by the cohesin complex and insulator factors. However, recent evidence also suggests a role for noncoding RNAs, such as enhancer-associated RNAs and promoter upstream transcripts, in shaping enhancer-promoter connectivity. While the nuclear RNA exosome, together with targeting complexes, poly(A) tail exosome targeting connection and nuclear exosome targeting complex, controls the decay of noncoding RNAs, it remains unclear whether these complexes regulate three-dimensional chromatin contacts. Chromatin recruitment maps of the nuclear exosome targeting complex subunit ZCCHC8, the poly(A) tail exosome targeting connection subunit ZFC3H1, and the RNA helicase MTR4 in human cells reveal that these factors associate with sites of enhancer-promoter interactions. Depletion of these factors leads to the accumulation of ncRNAs, notably enhancer-associated RNAs and promoter upstream transcripts, and increases cohesin occupancy at these sites. Chromatin conformation capture analysis reveals that MTR4 modulates long-range enhancer-promoter contacts. Upon loss of MTR4, enhancer-promoter contacts increase while intraloop contacts decrease, suggesting that MTR4 facilitates loop extrusion. These data highlight a key interplay between cohesin-mediated enhancer-promoter interactions and the regulation of noncoding RNAs by nuclear RNA exosome targeting complexes that is consistent with a role for RNA in genome folding.

Cohesins↗
PubMed1994

Anti-HCV seroconversion in multitransfused and immunocompromised patients. A long-term longitudinal study.

In a series of 6 multitransfused, immunocompromised patients, the diagnosis of posttransfusion hepatitis C was based upon the analysis of long-term follow-up serum samples. The HCV RNA was detected by a nested PCR assay using primers located in the 5' noncoding region (5'NCR), and anti-HCV antibodies were assayed with third-generation tests. The interval between the first rise in alanine aminotransferase and seroconversion varied from less than 5 months to more than 38 months. Five out of 6 patients seroconverted after 14 months or later. In most cases, the anti-NS3 and anti-NS4 antibodies appeared first. In such patients, the etiology of chronic liver disease may thus be overlooked for 1 or more years, a definitive diagnosis requiring the detection of HCV RNA.

Adult↗
PubMed1998

Epidemiological and clinical aspects of hepatitis G virus infection in blood donors and immunocompromised recipients of HGV-contaminated blood.

BACKGROUND AND OBJECTIVES: The infectiousness and clinical relevance of the newly discovered blood-borne Flaviviridae-like agent, termed hepatitis G virus (HGV), are not well understood. MATERIALS AND METHODS: Twenty-three transfusion recipients of two HGV-affected long-term blood donors were studied for HGV genome and antibodies to the putative envelope 2 glycoprotein (anti-E2) of HGV. Nine recipients had nonhematological disorders and 14 suffered from severe hematological diseases and 7 of them received allogeneic bone marrow or blood stem cell transplantation. The molecular epidemiology of the observed HGV infection was studied by direct sequencing of parts of the 5'-noncoding region, NS3, and NS5 region of HGV in the 2 long-term donors and in their 6 recipients who became HGV RNA positive. Additionally, 549 individuals-homologous (n = 254) and autologous blood donors (n = 202), and medical staff (n = 89)--were investigated for the presence of HGV RNA. RESULTS: HGV RNA in serum was found in 15 of the 23 (65%) transfusion recipients with known exposure of HGV-contaminated blood. Seven of the remaining 8 recipients showed only an anti-E2 response, indicating previous HGV infection with spontaneous clearance of the virus. In one recipient neither HGV RNA nor anti-E2 could be detected. Molecular evidence for HGV transmission by the 2 donors was found in 3 of the 6 recipients studied. The alanine aminotransferase levels were not significantly different in the HGV RNA positive and negative recipients, and none of the 23 recipients developed posttransfusion hepatitis. Persistent HGV infection was observed especially in recipients with severe hematological disorders or in those in whom intensive immunosuppressive treatment was necessary. Of the 549 individuals studied, 10 (1.8%) were healthy carriers of HGV RNA. CONCLUSION: The persistence of transfusion-acquired HGV infection is not associated with acute or chronic hepatitis, but may be influenced by the recipient's underlying disease.

Adult↗
PubMed1990

cis-acting sequences required for in vivo amplification of genomic RNA3 are organized differently in related bromoviruses.

Cowpea chlorotic mottle virus (CCMV) is a positive-strand RNA virus that infects dicotyledonous plants. The genome comprises three capped RNAs: RNA1 (3.2 kb), RNA2 (2.9 kb), and RNA3 (2.1 kb). cis-Acting sequences required for amplification in vivo were explored for RNA3, which does not contribute trans-acting factors to viral RNA replication. Using a CCMV cDNA expression system, deletions throughout RNA3 were constructed and tested for successful replication in barley protoplasts coinoculated with RNAs 1 and 2. As previously found for RNA3 of the related brome mosaic virus (BMV) (R. French and P. Ahlquist, 1987, J. Virol. 61, 1457-1465), either of the two coding regions can be individually deleted without blocking RNA3 amplification. However, in striking contrast to BMV, the entire intercistronic noncoding region separating these genes is also dispensable for CCMV RNA3 amplification. Moreover, although simultaneous deletions of the 3a and coat protein genes were deleterious for BMV RNA3 accumulation, CCMV RNA3 derivatives bearing larger deletions encompassing the 3a gene, intercistronic region, and coat protein gene amplify to high levels. Thus, unlike BMV RNA3, cis-acting sequences required for CCMV RNA3 amplification map solely in the 5' and 3' noncoding regions. Normal levels of CCMV RNA3 accumulation require over 125 but no more than 220 bases from the 3' noncoding region, and no more than the first 89 bases of the 238-base-long 5' noncoding region.

Chromosome Deletion↗
PubMed1991

HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts.

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.

Amino Acid Sequence↗
PubMed2025

Global lncRNA expression profiles in medulloblastoma reveal crucial lncRNA-oncogene interactions in Sonic hedgehog and Group 4.

BACKGROUND: Advances in multi-omic studies have improved medulloblastoma (MB) characterization, yet novel molecular biomarkers are needed to refine tumor biology and therapeutic strategies. Current profiling mainly targets the protein-coding genome, while the potential of noncoding regions remains unexplored. This study aims to identify long noncoding RNAs (lncRNAs), emerging as crucial regulators in MB, as potential key biomarkers specific to molecular group, enhancing understanding of MB's genomic landscape. METHODS: RNA-seq data from 54 Spanish MB patients (C1) and 207 public samples (C2) were analyzed to profile lncRNAs. Expression and Weighted Gene Coexpression Network (WGCNA) analyses were performed to identify lncRNA-oncogene interactions. Group-specific interactions were examined to infer their role in MB pathogenesis and highlight potential lncRNA involvement in disease mechanisms. RESULTS: LncRNA expression profiles identified 4 clusters corresponding to the MB molecular groups, confirming their potential as biomarkers. Expression and WGCNA analyses revealed group-specific lncRNAs for Sonic hedgehog (SHH), Group 3 (Gr3), and Group 4 (Gr4) MB. Lnc-SMARCA2 was exclusively upregulated in SHH MB, and associated with ATOH1 and PDLIM3, key cilium regulators of this group's cell of origin. In Gr4 MB, MGC32805 and LOC107986446 were upregulated and linked to SNCAIP, potentially influencing PRDM6 activation via enhancer hijacking. Additionally, a 5-lncRNA signature linked to phototransduction was exclusive to Gr3, offering insights into its lineage switch and molecular regulation. CONCLUSIONS: Lnc-SMARCA2 and, MGC32805 and LOC107986446, are exclusively deregulated in SHH and Gr4 MB, respectively, and directly associated with group-specific MB oncogenes, representing promising novel biomarkers and therapeutic targets in MB.

cancer biomarkers↗
PubMed1996

Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.

R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.

Animals↗
PubMed2001

Short report: genetic heterogeneity of Japanese encephalitis virus assessed via analysis of the full-length genome sequence of a Korean isolate.

We determined the full-length genome sequence of Japanese encephalitis virus (JEV) K94P05 isolated in Korea. Sequence analysis showed that the 10,963-nucleotide-long RNA genome of K94P05 was 13 or 14 nucleotides shorter than the genome of other JEV isolates because of a deletion in the 3' noncoding region of K94P05. Compared with sequences of other JEV isolates, the full-length nucleotide sequence showed 89.0-89.6% homology, and the deduced amino acid sequence showed between 96.4-97.3% homology. A region of approximately 60 nucleotides immediately downstream of the open reading frame stop codon of K94P05 showed high sequence variability as compared with other JEV isolates. K94P05 formed a distinct group within a phylogenetic tree established with the full-length genome sequences. Cross-neutralization studies showed that polyclonal antibodies to Korean isolates were 3 times better at neutralizing the Korean isolates than antibodies to Nakayama-NIH. These findings suggest that Korean JEV K94P05 is genetically and antigenically distinct from other Asian JEV isolates.

Amino Acid Sequence↗
PubMed1988

Sigma region located between C mu and C delta genes of human immunoglobulin heavy chain: possible involvement of tRNA-like structure in RNA splicing.

Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the mu-delta intron, two repeating units were contained. One 442-base-long fragment located JH-mu intron (defined as "sigma mu(sigma mu)") occupied the position in the mu-delta intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of C gamma gene was also found in the mu-delta intron. We defined the repeats in the mu-delta intron as "SIGMA (sigma)". The polarities of the longer repeats in the genome were opposite between the mu-delta intron and the upstreams of C gamma genes. These inverted copies (defined as sigma gamma 3 and sigma gamma 4), located 6 kb upstream of their respective C gamma's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig C gamma cluster.

Animals↗
PubMed2026

Long noncoding RNAs and diabetic retinopathy: Current understanding, future directions and challenges.

Diabetic retinopathy remains as the leading cause of preventable blindness in working-aged people. The pathophysiology of this sight-threatening disease is complex and involves intricate interactions among metabolic, hemodynamic and epigenetic pathways, leading to molecular, structural, functional and genomic abnormalities in retinal vascular and nonvascular cells. Diabetes also results in differential expressions of several noncoding RNAs, including micro RNAs (miRNAs) and long noncoding RNAs (LncRNAs). Compared to about 2000 miRNA identified in human genome thus far, more than 30,000 LncRNA transcripts have been already identified, but the function and mechanism of action of most of the LncRNAs is still not fully characterized, and there remains a possibility that some LncRNAs could have diverse functions under different contexts. LncRNAs are mainly noncoding, but they have many regulatory functions, and regulate gene expression by interacting with DNA, RNA and protein. Aberrant expressions of several LncRNAs including MALAT1, MEG3, HOTAIR, MIAT1 and H19, is associated with metabolic abnormalities implicated in the pathogenesis of diabetic retinopathy. LncRNAs are also released into circulation and show high organ and cell specificity, and greater disease-associated differences compared to disease-associated mRNAs. Furthermore, LncRNAs maintain stable expression in the plasma and can be isolated from total RNA present in biological samples including blood, which makes them promising and reliable candidates for diagnostic or prognostic markers and therapeutic targets for various diseases. With continued improvement in innovative RNA modifications and delivery modalities, use of LncRNAs as possible biomarkers, and of LncRNA-based therapeutics, for diabetic retinopathy appears promising.

Biomarkers↗
PubMed1991

Unregulated expression of the erythropoietin receptor gene caused by insertion of spleen focus-forming virus long terminal repeat in a murine erythroleukemia cell line.

A murine erythroleukemia (MEL) cell line, F5-5, expressed 10,000 binding sites for erythropoietin (EPO) per cell, 10-fold more than was expressed by other murine erythroleukemia cell lines and normal erythroid progenitors. Northern (RNA) and Southern blot analyses revealed overexpression of mRNA for the EPO receptor (EPOR) and rearrangement of one of the EPOR gene alleles in F5-5 cells, respectively. Molecular cloning of F5-5-derived cDNA encoding EPOR revealed that the 5' noncoding region of the EPOR cDNA corresponds to the 3' long terminal repeat sequence of the polycythemic strain of Friend spleen focus-forming virus (F-SFFVP). The aberrant EPOR transcripts containing the 3' long terminal repeat sequence were mainly expressed in F5-5 cells. The same integration upstream of the EPOR gene was also observed in other subclones and the parent cell line. It is possible that overexpression of EPOR by viral promoter insertion will confer growth advantage to an F-SFFVP-infected erythroid progenitor cell, leading to positive clonal selection through further leukemogenic steps.

Amino Acid Sequence↗
PubMed1995

Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome.

We report the complete nucleotide sequence of the M and the S genome segments and a portion of the L segments of two hantavirus isolates from Peromyscus maniculatus trapped in eastern California. The isolates, Convict Creek 107 and 74 (CC107 and CC74) are genetically similar to viruses known to cause hantavirus pulmonary syndrome in New Mexico. CC107 and CC74 each have an M segment consisting of 3696 nucleotides with a coding potential of 1140 amino acids in the virus complementary-sense RNA (cRNA). The S segments of CC107 and CC74 are 2083 and 2047 nucleotides long, respectively, and each has an ORF in the cRNA capable of encoding a protein of 428 amino acids. Unusually long 3' noncoding regions of 757 and 721 nucleotides follow the S segment ORF of CC107 and CC74, respectively, and include numerous imperfect repetitive sequences. Whereas the M and S segments of any given hantavirus typically appear to diverge at comparable rates from homologous genes of any other hantavirus, CC107 and CC74 have M segments that differ by only 1% from one another but S segments that differ by 13%. After trivial explanations are rendered improbable, i.e., by consideration of the genetics of closely and distantly related hantaviruses, the most likely explanation for our data is that hantavirus genome segment reassortment occurred within rodent populations in California.

Animals↗
PubMed1979

Structure of the constant and 3' untranslated regions of the murine gamma 2b heavy chain messenger RNA.

The complete coding sequence for the constant region of the mouse gamma 2b immunoglobulin heavy chain and the 3' untranslated region has been determined. The coding portion of the sequence is 1008 nucleotides long (amino acid residues 114 to 449), and the 3' noncoding region contains 102 nucleotides preceeding the polyadenylate. An extra carboxyl-terminal lysine residue which had not been observed in the gamma 2b or other gamma subclass protein sequences occurs in the nucleotide sequence and is probably processed posttranslationally. A 17-nucleotide sequence occurs with slight variation twice in CH1 and once in CH2 domains in the same relative location but with different translational phase. This sequence may be the site of crossover in a gamma 2b . gamma 2a heavy chain variant, an indication of possible recombinational activity of some kind.

Animals↗
PubMed2005

A systematic search for new mammalian noncoding RNAs indicates little conserved intergenic transcription.

BACKGROUND: Systematic identification and functional characterization of novel types of noncoding (nc)RNA in genomes is more difficult than it is for protein coding mRNAs, since ncRNAs typically do not possess sequence features such as splicing or translation signals, or long open reading frames. Recent "tiling" microarray studies have reported that a surprisingly larger proportion of mammalian genomes is transcribed than was previously anticipated. However, these non-genic transcripts often appear to be low in abundance, and their functional significance is not known. RESULTS: To systematically search for functional ncRNAs, we designed microarrays to detect 3,478 intergenic and intronic sequences that are conserved between the human, mouse, and rat genomes, and that score highly by other criteria that characterize ncRNAs. We probed these arrays with total RNA isolated from 16 wild-type mouse tissues. Among 55 candidates for highly-expressed novel ncRNAs tested by northern blotting, eight were confirmed as small, highly-and ubiquitously-expressed RNAs in mouse. Of the eight, five were also detected in rat tissues, but none were detected at appreciable levels in human tissues or cultured cells. CONCLUSION: Since the sequence and expression of most known coding transcripts and functional ncRNAs is conserved between human and mouse, the lack of northern-detectable expression in human cells and tissues of the novel mouse and rat ncRNAs that we identified suggests that they are not functional or possibly have rodent-specific functions. Our results confirm that relatively little of the intergenic sequence conserved between human, mouse and rat is transcribed at high levels in mammalian tissues, possibly suggesting a limited role for transcribed intergenic and intronic sequences as independent functional elements.

Animals↗
PubMed1989

Nucleotide sequence of a cDNA encoding rat protamine and the haploid expression of the gene during rat spermatogenesis.

The nucleotide sequence of a 342-base cDNA encoding the rat protamine has been determined. This insert, isolated from a rat testis cDNA library, encodes a polypeptide of 50 amino acids of which 29 are arginine 9 are cysteine and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 170 bases and 18 bases of the 5'-noncoding region. Hybridization of the protamine cDNA with the RNA prepared from testes of prepubertal and sexually mature rats revealed that protamine mRNA is first detectable as a 600 nucleotide long molecule in the 35-day old testis containing around 15% of round spermatids but not in testis of younger animals. The RNA of 50-day old and sexually mature rats was found to contain a second protamine mRNA which is around 500 nucleotides in length. Hybridization of the protamine cDNA with the RNA of isolated spermatids of the mature testis resulted in 2 prominent hybridization signals (600 and 500 bp) while the faint signal obtained with the RNA of pachytene spermatocytes (600 bp) was found to be due to contamination of the cell preparation by spermatids. After digestion of the mRNAs with ribonuclease H a single hybridization band even smaller than 500 nucleotides was obtained. As demonstrated on testis sections the transcripts are confined to the central layers of the tubuli seminiferi corresponding to the spatial arrangement of corresponding to the spatial arrangement of postmeiotic cells. The results indicate that the protamine gene in the rat is postmeiotically expressed and that the mRNA undergoes post-transcriptional processing that includes a reduction in molecular size with respect to the poly-(A)+ tail.

Amino Acid Sequence↗
PubMed1985

Complete sequence of ovine alpha s2-casein messenger RNA.

The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein.

Amino Acid Sequence↗
PubMed2005

Genome-wide analyses of two families of snoRNA genes from Drosophila melanogaster, demonstrating the extensive utilization of introns for coding of snoRNAs.

Small nucleolar RNAs (snoRNAs) are an abundant group of noncoding RNAs mainly involved in the post-transcriptional modifications of rRNAs in eukaryotes. In this study, a large-scale genome-wide analysis of the two major families of snoRNA genes in the fruit fly Drosophila melanogaster has been performed using experimental and computational RNomics methods. Two hundred and twelve gene variants, encoding 56 box H/ACA and 63 box C/D snoRNAs, were identified, of which 57 novel snoRNAs have been reported for the first time. These snoRNAs were predicted to guide a total of 147 methylations and pseudouridylations on rRNAs and snRNAs, showing a more comprehensive pattern of rRNA modification in the fruit fly. With the exception of nine, all the snoRNAs identified to date in D. melanogaster are intron encoded. Remarkably, the genomic organization of the snoRNAs is characteristic of 8 dUhg genes and 17 intronic gene clusters, demonstrating that distinct organizations dominate the expression of the two families of snoRNAs in the fruit fly. Of the 267 introns in the host genes, more than half have been identified as host introns for coding of snoRNAs. In contrast to mammals, the variation in size of the host introns is mainly due to differences in the number of snoRNAs they contain. These results demonstrate the extensive utilization of introns for coding of snoRNAs in the host genes and shed light on further research of other noncoding RNA genes in the large introns of the Drosophila genome.

Animals↗