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PubMed2004

Systematic identification of abundant A-to-I editing sites in the human transcriptome.

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

Adenosine↗

PubMed2006

The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation.

The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection.

Animals↗

PubMed1993

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↗

PubMedDate not supplied

Stimulation of transcription of the yeast tRNATyr gene in cell-free extracts by tyrosyl-tRNA synthetase.

Eukaryotic transfer RNA genes have two internal discontinuous control regions, the A and B blocks, which correspond approximately to the D-stem and the pseudo-U arm of tRNA. In reconstituted transcription systems at least two components are required to direct accurate initiation by RNA polymerase (refs 4,5). However, little is known about the mechanism of interaction of the internal promoter sequences with factors and RNA polymerase C within the transcription complex, although tRNA-like conformation of the B block sequence was surmised to be critical for DNA recognition. By analogy with the 5S RNA system, where a transcription factor required for 5S DNA expression was shown to interact both with 5S RNA and with the noncoding strand of the 5S gene, we explored the possibility that a protein which normally binds to tRNA could also interact with the tRNA gene and regulate its transcription. Here we show that in vitro transcription of the yeast SUP4 tRNATyr gene in crude yeast extracts is strongly stimulated by tyrosyl-tRNA synthetase (TyrRS) but not by two other non-cognate synthetases. Substrates of the synthetase, tRNATyr and tyrosine, interfere with stimulation of tRNA synthesis.

Amino Acyl-tRNA Synthetases↗

PubMed1989

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions.

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.

Amino Acid Sequence↗

PubMed2026

Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation.

Long noncoding RNAs are emerging as critical regulators of acute kidney injury (AKI). In this study, the pathologic role of pseudogene-derived long noncoding RNAs GSTM3P1 (human)/Gstm2-ps1 (mouse) in sepsis-associated AKI (SA-AKI) was investigated. Glutathione S-transferase mu 3, pseudogene 1 (GSTM3P1)/glutathione S-transferase mu 2, pseudogene 1 (Gstm2-ps1) were transiently up-regulated in kidney proximal tubular cells at the early stage of SA-AKI in mice treated with lipopolysaccharide (LPS) or cecal ligation and puncture, as well as in LPS-treated proximal tubular cells. Functionally, overexpression of GSTM3P1/Gstm2-ps1 exacerbated LPS-induced proximal tubular cell apoptosis and oxidative stress. In contrast, proximal tubule-specific Gstm2-ps1 knockout mice were significantly protected from LPS-induced AKI, as evidenced by improved renal function and reduced apoptosis, kidney injury markers, and reactive oxygen species. Similarly, these mice showed renal protective effects against cecal ligation and puncture-induced AKI. Mechanistically, overexpression of GSTM3P1/Gstm2-ps1 in proximal tubular cells markedly suppressed parent gene GSTM3/GSTM2 protein but not mRNA expression, indicating a translational repression. Restoration of GSTM3/GSTM2 rescued proximal tubular cells from LPS-induced apoptosis. Furthermore, an RNA pulldown assay revealed that Gstm2-ps1 binds to human antigen R (HuR), a known post-transcriptional regulator for mRNA stability and translation. Overexpression of HuR antagonized Gstm2-ps1-mediated repression of GSTM2, associated with increased cell survival after LPS injury. In conclusion, the early induction of GSTM3P1/Gstm2-ps1 in SA-AKI exacerbates kidney injury by a novel mechanism to sequester HuR and inhibit the translation of parent gene GSTM3/gstm2 for oxidative stress detoxification.

Animals↗

PubMed2004

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Amino Acid Sequence↗

PubMed1988

A retroviral RNA secondary structure required for efficient initiation of reverse transcription.

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.

Animals↗

PubMed1998

Interference with Physalis mottle tymovirus replication and coat protein synthesis by transcripts corresponding to the 3'-terminal region of the genomic RNA--role of the pseudoknot structure.

The role of the 3' noncoding (NC) region of Physalis mottle tymovirus genomic RNA in the multiplication of the virus was examined using an in vivo protoplast assay system. Coat protein (CP) synthesis was specifically inhibited by sense 3' NC region transcripts. To establish the role of the pseudoknot structure present in the NC region in virus multiplication, four site-specific mutants, two of which disrupted the pseudoknot structure while the other two restored the structure, were constructed. Interestingly, none of the four sense mutant transcripts inhibited CP synthesis, suggesting that the specific sequence representing the 3' terminal pseudoknot structure may play an important role in virus multiplication. However, the wild-type antisense 3' NC transcript as well as the four antisense mutant transcripts inhibited CP synthesis, suggesting that the inhibitions by antisense transcripts could be due to the formation of RNA-RNA hybrids at the 3' end of the genomic RNA.

Capsid↗

PubMed1997

Inhibition of influenza viral polymerases by minimal viral RNA decoys.

All gene segments of influenza virus share a common feature at their respective termini. Both the 5'- and 3'-terminal sequences are highly conserved and possess partial inverted complementarity. This allows for the formation of a double-stranded duplex, which plays a major role in transcription, replication and packaging of the viral genome. In vitro studies have shown that the viral polymerase binds to short RNA molecules containing these termini. In this study, attempts were made to test whether mini-RNA decoys containing either or both termini can inhibit the activity of the viral polymerase in vivo. RNA molecules containing either the 5' or the 3' noncoding sequences were unable to inhibit NS-CAT RNA replication, while mini-RNA decoys consisting of both the 5' and 3' noncoding sequences of vRNA or cRNA were able to efficiently inhibit the activity of the viral polymerases expressed from vaccinia virus vectors.

Base Sequence↗

PubMed2019

Distinct types of short open reading frames are translated in plant cells.

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Bryopsida↗

PubMed2025

Expression of long noncoding RNAs in peripheral blood mononuclear cells of patients with type 1 diabetes mellitus: potential biomarkers for disease onset.

OBJECTIVE: Long non-coding RNAs (lncRNAs) do not encode proteins and are transcripts longer than 200 nucleotides. The precise involvement of lncRNAs in type 1 diabetes mellitus (T1DM) pathogenesis remains unclear. Therefore, this study aimed to analyze the expressions of five lncRNAs in peripheral blood mononuclear cells of individuals with T1DM and without DM. MATERIALS AND METHODS: This study comprised 27 patients with T1DM (cases) and 13 individuals without DM (controls). The case group was divided into two subgroups based on T1DM duration: < 5 years of diagnosis group and long-term diabetes group (≥5 years). LncRNA expression was evaluated by qPCR. RESULTS: MALAT1 and TUG1 were upregulated in patients within the first five years of diagnosis of T1DM compared to the other groups. MEG3 was upregulated in the case group of < 5 years of diagnosis compared to controls. TUG1 and MALAT1 levels were negatively correlated with the duration of T1DM, while TUG1 and MEG3 were positively correlated with glycated hemoglobin levels. Bioinformatics analysis revealed that MALAT1, MEG3, and TUG1 regulate and interact with protein-codifying genes and microRNAs involved in T1DM-related pathways. CONCLUSION: Our study revealed MALAT1, MEG3, and TUG1 upregulation in patients within the first five years of diagnosis of T1DM.

Humans↗

PubMed2001

Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex.

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5'- and 3'-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3'-end and at nucleotides 3', 6' or 8' at the 5'-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6' contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3' and 8' at the 5'-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.

DNA-Directed RNA Polymerases↗

PubMed1993

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats.

By Northern blot analysis with the digoxigenin-labeled antisense RNA probes of the noncoding regions, the transcripts of three calmodulin (CaM) genes, CaMI, CaMII, and CaMIII, were separately detected in 12 different tissues of adult Wistar albino rats, without any cross-hybridization. The mRNAs of all three CaM genes were abundant in the central nervous system (CNS) as well as in the testis, although ubiquitous expression was detected at low levels in the other tissues. There were subtle but significant differences in the tissue-specific distribution of the three CaM gene RNAs. By in situ hybridization, strong hybridization of the three CaM gene probes was observed in common in large projection neurons of the CNS: the hippocampal pyramidal cells, the cerebellar Purkinje cells, and the large neurons of the cerebral neocortex, the pyriform cortex, the mesencephalon, the pons, and the spinal cord. The expression of the three CaM genes was at lower levels in small interneurons of the CNS. These profiles of expression were almost the same among the three CaM genes. Thus, all three CaM genes were coordinately expressed in neurons of the adult rat CNS. Certain regulatory mechanisms of the three CaM genes seemed to mediate similar tissue- and cell type-specific expression in the CNS.

Animals↗

PubMed1987

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.

Base Sequence↗

PubMed2006

Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae.

Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.

Aptamers, Nucleotide↗

PubMed1995

Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region.

The ie-1 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a transregulatory protein (IE1) which accelerates the expression of early and late virus genes. Transcription of ie-1 occurs immediately upon infection from a conserved CAGT motif and continues into the late phases. To examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed mutagenesis. The ie-1 upstream activating region, extending from nucleotide -546 to the TATA element at -34, stimulated ie-1 basal promoter activity more than 1,000-fold when transfected into uninfected Spodoptera frugiperda SF21 cells. However, when introduced into the genome of AcMNPV recombinants, the ie-1 upstream activating region had only a minimal twofold effect early in infection. Instead, maximum steady-state levels of early ie-1 RNAs required sequences within the 5' noncoding leader region extending from +11 to +24 relative to the RNA start site (+1). The +11 to +24 noncoding region did not influence the stability of ie-1 transcripts. When assayed by in vitro transcription, deletion of the +11 to +24 region reduced the levels of ie-1 runoff RNAs. Thus, this downstream activating sequence controlled the rate of early ie-1 transcription. A larger overlapping region from +11 to +36 affected steady-state levels of ie-1 RNAs late (24 h) in infection. Deletion of sequences that included the conserved CAGT start site abolished early ie-1 transcription. Thus, ie-1 is the first example of an early baculovirus gene in which essential cis-acting regulatory elements reside within the 5' noncoding region and include sequences comprising the RNA start site.

Animals↗

PubMed1988

An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

Animals↗