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Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms.

The complete nucleotide sequence of peanut clump virus RNA 2 has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 4503 residues in length and contains six long open reading frames (ORFs). The cistron for the coat protein (ORF 1) is located nearest the 5' end of the RNA and is separated from the 5' extremity by a noncoding region of 389 nucleotides. ORF 2 starts one nucleotide upstream of the first residue of the UGA termination codon of the coat protein cistron and encodes a polypeptide of 39300 M(r). Four additional ORFs with the potential to encode polypeptides with approximate M(r)'s of 51,000 (ORF 3), 13,800 (ORF 4), 9400 (ORF 5), and 6800 (ORF 6) are found on the 3' proximal half of the RNA. ORFs 3, 4, and 5 resemble the Triple Gene Block found in potexviruses, carlaviruses, a hordeivirus, and a furovirus. The 276 nucleotide 3' noncoding region of RNA 2 displays extensive sequence homology with the 3' noncoding region of RNA 1. Several peanut clump virus isolates propagated on Nicotiana benthamania contain RNA 2's significantly shorter than that of the type isolate. Partial characterization of two such isolates reveals that their RNA 2's have undergone deletions within ORF 2.

Amino Acid Sequence↗

In vivo transcription of the 5'-terminal extracistronic region of vesicular stomatitis virus RNA.

In vivo transcription and polyadenylation at the junction of the L cistron and the 5'-terminal extracistronic region of vesicular stomatitis virus RNA was investigated. Annealing of 5'32P-labeled RNA representing the 5'-terminal noncoding 77 nucleotides of vesicular stomatitis virus genomic RNA to L gene mRNA resulted in specific duplex formation. Two specific RNase T1- and RNase A resistant duplexes, 66 and 77 nucleotides long, bound to oligodeoxythymidylic acid cellulose. The specific sizes of the duplexes and their selection by oligodeoxythymidylic acid cellulose chromatography demonstrated that they were covalently linked to the polyadenylic acid tail of L gene mRNA. These data strongly suggest that the viral polymerase polyadenylates L gene mRNA in vivo by using the stretch of seven uridine residues at the end of the L cistron and that the polymerase can resume transcribing the 5'-terminal extracistronic region, resulting in a covalent linkage of the transcript to the polyadenylic acid tail of L gene mRNA.

Genes↗

Identification and characterization of human BIC, a gene on chromosome 21 that encodes a noncoding RNA.

BIC was originally identified as a gene transcriptionally activated by promoter insertion at a common retroviral integration site in B cell lymphomas induced by avian leukosis virus (Tam et al., Mol. Cell. Biol. 17 (1997) 1490). The human homolog of this gene was cloned and characterized. It consists of three exons within a 13 kb region located in chromosome 21q21. Similar to the avian homolog, the human BIC lacks a long open reading frame (ORF). Highest levels of BIC expression are detected in the spleen and thymus by Northern analysis. In addition, the mouse homolog of BIC was identified. Comparison of BIC cDNAs from human, mouse and chicken reveals 78% identity over 138 nucleotides. However, there is no homology among the multiple short ORFs present in these cDNAs. The region of sequence homology is predicted by computer analysis to form an imperfect RNA duplex, which is structurally similar among the three species. Based on the lack of a conserved ORF and the evolutionary conservation of RNA secondary structure, we presume that BIC functions as a noncoding RNA.

Animals↗

Rat pancreatic ribonuclease messenger RNA. The nucleotide sequence of the entire mRNA and the derived amino acid sequence of the pre-enzyme.

We have cloned via recombinant DNA technology the mRNA sequence of rat pancreatic ribonuclease, and have determined the entire nucleotide sequence of the mature message. Clones bearing RNase sequences within a double-stranded complementary DNA library of rat pancreatic mRNA were initially detected by hybridization with size-fractionated rat pancreatic polyadenylated RNA that included mRNA 0.85 to 1.0 kilobase in length. Recombinant plasmids bearing RNase mRNA sequences were conclusively identified by comparison of the amino acid sequence of the encoded protein with the known amino acid sequence of rat RNase. RNase mRNA is 783 nucleotides in length, plus a poly(a) tail with an average length of 140 nucleotides, and contains long 5' and 3' noncoding regions relative to other pancreatic mRNAs. It encodes a secretory preRNase of 152 amino acid residues including a signal peptide of 25 amino acids.

Amino Acid Sequence↗

The diagnosis and therapy of brain tumours.

Neoplasms arising from glial cells make up the most common group of primary brain tumors. The clinical outcome, especially the survival rates of the patients with brain tumours depend on tumour grade expressing its malignancy. A prognosis for glioblastomas (WHO IV) is very poor, but for astrocytomas (WHO I and II) it is relatively favourable. For oligodendrogliomas a longer survival time than for glioblastomas is observed. There is evidence that oxidative stress and reactive oxygen species (ROS) are crucial in the etiology and progression of a number of human diseases, including neoplasms. An oxidative damage of DNA, lipids and proteins is caused mainly with hydroxyl radical (*OH), the most reactive ROS species and may be seriously deleterious. In addition to all four basic nucleotides: adenosine (A), guanosine (G), tymidine (T) and cytosine (C), 5-methylcytosine (m5C) is a rare but normal component of cellular DNA and occurs mainly within a sequence of a structural gene or in regulatory regions. In the reaction with hydroxyl radical all DNA components can be modified, but m5C is relatively easily deaminated to thymine, which, in turn, pairs with adenine and after a round of replication, CG to TA transition occurs. Because thymine is a normal DNA base, therefore the product of spontaneous deamination of m5C is not so easily detected by cell's DNA repair system. Thus, 5-methylcytosine residue constitutes a mutational hotspot and DNA methylation pattern in patients might be useful as a primary diagnostic tool or as a marker for early detection of relapse of the disease. In recent years a new mechanism of posttranscriptional gene silencing has been discovered and named RNA interference (RNAi). This phenomenon is based on mRNA degradation mediated by small double-stranded RNA molecules, approximately 19-28 nucleotides in length, called short interfering RNAs (siRNAs). These molecules are produced from long dsRNAs by a dsRNA-specific endonuclease (DICER) and form 300 kD multi-enzyme complex (RISC) which by Watson-Crick base-pairing of noncoding strand with their mRNA-targets induce specific degradation. The high sequence-specificity of RNAi makes it a new, promising tool in a gene-function analysis as well as in potential therapeutics development.

Animals↗

SINEs and LINEs: the art of biting the hand that feeds you.

SINEs and LINEs are short and long interspersed retrotransposable elements, respectively, that invade new genomic sites using RNA intermediates. SINEs and LINEs are found in almost all eukaryotes (although not in Saccharomyces cerevisiae) and together account for at least 34% of the human genome. The noncoding SINEs depend on reverse transcriptase and endonuclease functions encoded by partner LINEs. With the completion of many genome sequences, including our own, the database of SINEs and LINEs has taken a great leap forward. The new data pose new questions that can only be answered by detailed studies of the mechanism of retroposition. Current work ranges from the biochemistry of reverse transcription and integration invitro, target site selection in vivo, nucleocytoplasmic transport of the RNA and ribonucleoprotein intermediates, and mechanisms of genomic turnover. Two particularly exciting new ideas are that SINEs may help cells survive physiological stress, and that the evolution of SINEs and LINEs has been shaped by the forces of RNA interference. Taken together, these studies promise to explain the birth and death of SINEs and LINEs, and the contribution of these repetitive sequence families to the evolution of genomes.

Animals↗

MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.

Base Sequence↗

The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus.

The 15,697-nucleotide sequence of Paracentrotus lividus mitochondrial DNA is reported. This genome codes for 2 rRNAs, 22 tRNAs, and 12 mRNAs which specify 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement differs from that of other animal species. The two ribosomal genes 16 S and 12 S are separated by a stretch of about 3.3 kilobase pairs which contains the ND1 and ND2 genes and a cluster of 15 tRNA genes. The ND4L coding sequence is not contained in the ND4 mRNA but has its own mRNA which maps between the tRNA(Arg) and the Co II genes. The main noncoding region, located in the tRNA gene cluster, is only 132 nucleotides long, but contains sequences homologous to the mammalian displacement loop. Other short noncoding sequences are interspersed in the genome: they contain a conserved AT consensus which probably has a role in transcription or RNA processing. As regards the mitochondrial genetic code, the codons AGA and AGG specify serine and are recognized by a tRNA with a GCU anticodon, whereas AUA and AAA code for isoleucine and asparagine rather than for methionine and lysine. Except for ND4L which starts with AUC and ATPase 8 which starts with GUG, AUG is used as the initiation codon. In 11 out of 13 cases the genes terminate with the canonical stop codons UAA or UAG. These observations suggest that during invertebrate evolution each lineage developed its own mechanism of mitochondrial DNA replication and transcription and of RNA processing and translation.

Amino Acid Sequence↗

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.

Amino Acid Sequence↗

5'-terminal nucleotide sequence of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The complete 5'-terminal nucleotide sequence of the MRNA coding for the bovine common precursor of corticotropin and beta-lipotropin has been determined. The 5'-32P-labelled, 21-nucleotides-long, single-stranded DNA fragment complementary to a portion of the 5'-noncoding region of the mRNA was prepared from a cDNA clone and elongated by reverse transcriptase reaction with the mRNA as template. The DNA transcript formed was sequenced by the procedure of Maxam and Gilbert, and the resultant sequence was cross-checked by two-dimensional electrophoretic analysis of the partial alkaline digest of the 5'-32P-labelled mRNA. The 5'-terminal nucleotide residue was determined by two-dimensional thin-layer chromatography of the complete hydrolysis product of the 5'-32P-labelled mRNA. The nucleotide sequence determined, which partially overlaps the known sequence of the cloned cDNA, reveals the complete 5'-terminal sequence of the mRNA. This, in conjunction with our previous data, defines the complete primary structure of the mRNA. The mRNA is composed of 1098 nucleotides, including an unusually long 5'-noncoding sequence of 128 nucleotides. The presence of a 'cap' structure at the 5' terminus of the mRNA is suggested. The 5'-terminal 48 nucleotide residues of the mRNA are extremely purine-rich, having an A + G content of 83%, whereas all pyrimidine-rich segments are located downstream from there. Because the 5'-noncoding region of the mRNA contains three segments of potential secondary structure which partially overlap, it can exist in a number of alternative base-pairing configurations. However, its interaction with the 3'-terminal segment of 18-S rRNA at the site of maximal complementarity would fix the mRNA configuration in such a way as to bring the possible site of ribosome binding near the initiation codon.

Adrenocorticotropic Hormone↗

NAT10 is critical to block RNA sensing-induced IFN-β transactivation in viral infection.

UNLABELLED: Cells detect invading viruses and produce type I interferons (IFNs) to stimulate an innate antiviral effector response. However, IFN levels must be fine-tuned to achieve antiviral efficacy while limiting hyperinflammatory and tissue-damaging effects. Here, we report that NAT10, a histone and cytidine acetyltransferase, regulates the production of type I IFNs and RNA virus infections. Depletion of NAT10 increased the expression of IFN-β and IFN-stimulated genes, and correspondingly impaired viral replication. Mechanistically, NAT10 dynamically associated with the IFN-β promoter and also negatively regulated IRF3's chromatin associations through modulation of long noncoding RNAs that inhibit IRF3. Treatment of cells with Remodelin, a NAT10 inhibitor, similarly increased IFN-β expression and inhibited viral infections. Overall, our findings reveal NAT10 is a potential host-directed target for antiviral treatment via regulation of type I IFN. IMPORTANCE: Type I interferons (IFNs) signaling pathway is critical to cellular defense and innate immunity against evading pathogens, including viruses. However, induction of type I IFNs is fine-tuned to achieve the antiviral consequence while maintaining host cellular homeostasis. This paper presents a novel mechanism for the NAT10 protein to silence IFN-β induction through modulation of IRF3 activity at the promoter of IFN-β, and further demonstrates the therapeutic potential of the NAT10 inhibitor Remodelin to restrict viral infection while inducing IFN-β.

Interferon-beta↗

Human U19 intron-encoded snoRNA is processed from a long primary transcript that possesses little potential for protein coding.

While exons were originally defined as coding regions of split eukaryotic genes, introns have long been considered as mainly noncoding "genetic junk." However, recognition that a large number of small nucleolar RNAs (snoRNAs) are processed from introns of pre-mRNAs demonstrated that introns may also code for functional RNAs. Moreover, recent characterization of the mammalian UHG gene that encodes eight box C/D intronic snoRNAs suggested that some genes generate functional RNA products exclusively from their intron regions. In this study, we show that the human U19 box H/ACA snoRNA, which is encoded within the second intron of the U19H gene, represents the only functional RNA product generated from the long U19H primary transcript. Splicing of the U19H transcript, instead of giving rise to a defined RNA, produces a population of diverse U19H RNA molecules. Although the first three exons of the U19H gene are preserved in each processed U19H RNA, the 3' half of the RNA is generated by a series of apparently random splicing events. Because the U19H RNA possesses limited potential for protein coding and shows a predominant nucleoplasmic localization, we suggest that the sole function of the U19H gene is to express the U19 intronic snoRNA. This suggests that, in marked contrast to our previous dogmatic view, genes generating functionally important RNAs exclusively from their intron regions are probably more frequent than has been anticipated.

Alternative Splicing↗

Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA.

To distinguish the inhibitory effect of anti-sense RNA on translation from the effect on splicing, a plasmid (pLC32) was constructed from a cDNA clone of the Rous sarcoma virus (RSV) envelope gene (env) mRNA. Transcription of this plasmid results in the synthesis of RNA identical to the RSV env gene mRNA which does not require splicing to be expressed. Plasmids derived from pLC32 were also constructed in which the env gene coding sequence and 5' noncoding leader sequences were inserted in the opposite orientation relative to the RSV long terminal repeats (LTRs). pLC32 DNA transfected by the calcium phosphate coprecipitation technique efficiently rescued infectious virus from quail cells infected with an RSV mutant deleted in the env gene [R(-)Q cells], indicating that the intron sequences are dispensable in env gene expression. When the inverted constructs were cotransfected with pLC32, significantly less infectious virus was produced. The extent of the inhibition depended upon the concentration ratio of the two plasmids. The maximum inhibition (80%) occurred when the ratio of inverted constructs to pLC32 was 12:1. The inhibition is specific for the inverted orientation since cotransfection of pLC32 with several other plasmids containing viral LTRs and defective src and env genes at similar concentrations did not inhibit the production of infectious virus. In addition, the inverted constructs did not interfere with the expression of an LTR-driven chloramphenicol acetyltransferase gene. When cotransfected with a wild-type Prague A RSV DNA plasmid (pJD100), the inverted constructs also greatly inhibited expression and replication of virus in R(-)Q quail cells. These data suggest that the specific inhibition is caused by hybridization of complementary RNA transcribed from the inverted constructs to the env mRNA, thereby blocking its expression. The fact that expression of both intron-containing and intronless clones are inhibited to the same extent suggest that inhibition by anti-sense RNA from the env exon regions does not act at the level of RNA splicing.

Animals↗

C-ski cDNAs are encoded by eight exons, six of which are closely linked within the chicken genome.

The c-ski locus extends a minimum of 65 kb in the chicken genome and is expressed as multiple mRNAs resulting from alternative exon usage. Four exons comprising approximately 1.5 kb of cDNA sequence have been mapped within the chicken c-ski locus. However, c-ski cDNAs include almost 3 kb of sequence for which the exon structure was not defined. From our studies using the polymerase chain reaction and templates of RNA and genomic DNA, it is clear that c-ski cDNAs are encoded by a minimum of eight exons. A long 3' untranslated region is contiguous in the genome with the distal portion of the ski open reading frame such that exon 8 is composed of both coding and noncoding sequences. Exons 2 and 3 are separated by more than 25 kb of genomic sequence. In contrast, exons 3 through 8, representing more than half the length of c-ski cDNA sequences, are closely linked within 10 kb in the chicken genome.

Animals↗

Expression of a histone H1-like protein is restricted to early Xenopus development.

Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone H1, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone H1 mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone H1 3' noncoding consensus sequence involved in RNA processing.

Amino Acid Sequence↗

Human beta-globin messenger RNA. I. Nucleotide sequences derived from complementary RNA.

Sequence analysis studies were carried out on human beta-globin mRNA (beta-mRNA) prepared from alpha-thalassemic, sickle cell, and Hb A reticulocytes. Highly purified beta-mRNA served as substrate for the preparation of cDNA by RNA-dependent DNA polymerase. The cDNA was transcribed by Escherichia coli RNA polymerase and the resulting cRNA was analyzed. Over 300 nucleotides were assigned to the beta-mRNA coding region and 37 nucleotides were assigned to the 3'-terminal noncoding region. The normal termination codon is UAA which is separated by 28 nucleotides from an out of phase UAA triplet. The origin of each of the abnormally long beta-globin variants Tak and Cranston is consistent with reduplication of dinucleotides prior to the normal termination codon, and both globin variants can terminate at the out of phase UAA.

Anemia, Sickle Cell↗

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.

Amino Acid Sequence↗

Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses.

Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which constitute a separate serological group within the picornavirus family. Persistent TMEV infection in mice provides a relevant experimental animal model for the human demyelinating disease multiple sclerosis. To provide information about the TMEV classification, genome organization, and protein processing map, we determined the complete nucleotide sequence of the TMEV genome and deduced the amino acid sequence of the polyprotein coding region. The RNA genome, which is typical of the picornavirus family, is 8,098 nucleotides long. The 5' untranslated region is 1,064 nucleotides long (making it the longest in the picornavirus family after the aphthoviruses) and lacks a poly(C) tract. Computer-generated comparison of the 5' and 3' noncoding regions and polyprotein revealed the highest level of nucleotide and predicted amino acid identity between the TMEV and the cardioviruses encephalomyocarditis virus (EMCV) and Mengo virus. The TMEV polyprotein, which appears to be processed like EMCV since the amino acids flanking the putative proteolytic cleavage sites have been conserved, begins with a short leader peptide followed by 11 other gene products in the standard L-4-3-4 picornavirus arrangement. Because of these similarities, we propose that the TMEV be grouped with the cardioviruses. However, since TMEV and EMCV have different biophysical properties and show no cross-neutralization, they most likely belong in a separate cardiovirus subgroup.

Amino Acid Sequence↗