Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.
Mol Cell Biol 1990 Aug
PMID:N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts. 169 23

Erythropoiesis in vertebrates is characterized by sequential changes in erythropoietic site, erythroblast morphology, and hemoglobin synthesis. We have examined the expression of globin chains and the major erythroid transcription factor GATA-1 (previously known as GF-1/NF-E1/Eryf 1) from days 7.5 to 17.5 of mouse development. mRNAs for embryonic (epsilon y2, beta H1, and zeta) and adult (alpha and beta) globin chains were quantitated by RNase protection assays. Switching of globins within the alpha-globin cluster (alpha and zeta) was not strictly coordinated with that within the beta-globin cluster (epsilon y2, beta H1, and beta). Regulation of globin switches during development was primarily transcriptional. Of particular note, we found two developmental switches (beta H1 to epsilon y2 and epsilon y2 to beta) in the mouse, more analogous than previously thought to shifts found in human development. The erythroid transcription factor GATA-1, believed to be a principal regulator of genes expressed in erythroid cells, first appeared in the embryo in yolk sac at the time of blood island formation and remained at a low level during embryonic erythropoiesis (8 to 11 days) relative to that found later in fetal liver (12 to 15 days). The rise in GATA-1 mRNA in fetal liver paralleled and preceded the rapid accumulation of adult beta-globin RNA. RNase protection assays and a GATA-1-specific peptide antiserum were used to establish that a single GATA-1 polypeptide is expressed throughout mouse development. Overall, these findings suggest that the levels of this erythroid transcription factor during development may contribute to the differential gene activation characteristic of definitive versus primitive erythropoiesis.
Mol Cell Biol 1990 Dec
PMID:Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. 170 Oct 19

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.
Mol Cell Biol 1991 Mar
PMID:The tumor suppressor p53 is bound to RNA by a stable covalent linkage. 170 9

Two lyn proteins of 56 and 53 kDa have been observed in immunoprecipitates from a variety of murine and human cell lines and tissues. We report the cloning and nucleotide sequence of two distinct murine lyn cDNAs isolated from an FDC-P1 cDNA library. One of the cDNAs, designated lyn11, encodes a protein of 56 kDa which shares 96% similarity with human lyn. The other cDNA, designated lyn12, encodes a protein of 53 kDa. The proteins differ in the presence or absence of a 21-amino-acid sequence located 24 amino acids C terminal of the translational initiation codon. Using RNase protection analysis, we have identified mRNAs corresponding to both cDNAs in murine cell lines and tissues. Sequence analysis of murine genomic clones suggests that the distinct mRNAs are alternatively spliced transcripts derived from a single gene. Expression of both cDNAs in COS cells leads to the production of lyn proteins with the same molecular weight as the two forms of lyn proteins immunoprecipitated from extracts of FDC-P1 cells and mouse spleen. Subcellular fractionation studies and Western immunoblotting analysis suggest that both isoforms of lyn are membrane associated. The association of both lyn isoforms with the membrane fraction supports the notion that lyn, like other src-related kinases, may interact with the intracellular domain of cell surface receptors.
Mol Cell Biol 1991 Jul
PMID:Alternatively spliced murine lyn mRNAs encode distinct proteins. 171 Jul 66

Sex-lethal (Sxl), a key sex determination gene in Drosophila melanogaster, is known to express a set of three early transcripts arising during early embryogenesis and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs were distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). We have now analyzed the structures of the late Sxl transcripts by cDNA sequencing, Northern (RNA) blotting, primer extension, and RNase protection. The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. Besides the presence of the male-specific exon, no additional sex-specific splicing events were detected, although a number of non-sex-specific splicing variants were observed. In females, the various forms of late Sxl transcript potentially encode up to six slightly different polypeptides. All of the protein-coding differences occur outside the previously defined ribonucleoprotein motifs. One class of Sxl mRNAs also includes a second long ORF in the same frame as the first ORF but separated from it by a single ochre codon. The function of this second ORF is unknown. Significant amounts of apparently partially processed Sxl RNAs were observed, consistent with the hypothesis that the regulated Sxl splices occur relatively slowly.
Mol Cell Biol 1991 Jul
PMID:The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides. 171 Jul 69

The mammalian signal recognition particle (SRP) is a small cytoplasmic ribonucleoprotein required for the cotranslational targeting of secretory proteins to the endoplasmic reticulum membrane. The heterodimeric protein subunit SRP9/14 was previously shown to be essential for SRP to cause pausing in the elongation of secretory protein translation. RNase protection and filter binding experiments have shown that binding of SRP9/14 to SRP RNA depends solely on sequences located in a domain of SRP RNA that is strongly homologous to the Alu family of repetitive DNA sequences. In addition, the use of hydroxyl radicals, as RNA-cleaving reagents, has revealed four distinct regions in this domain that are in close contact with SRP9/14. Surprisingly, the nucleotide sequence in one of these contact sites, predicted to be mostly single stranded, was found to be extremely conserved in SRP RNAs of evolutionarily distant organisms ranging from eubacteria and archaebacteria to yeasts and higher eucaryotic cells. This finding suggests that SRP9/14 homologs may also exist in these organisms, where they possibly contribute to the regulation of protein synthesis similar to that observed for mammalian SRP in vitro.
Mol Cell Biol 1991 Aug
PMID:Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution. 171

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.
Mol Cell Biol 1991 Aug
PMID:Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5' exons and possible mechanism for the genesis of the 3' end of v-src. 171 5

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
J Mol Biol 1991 Aug 05
PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

In vitro transcripts from the 3' flanking regions of mustard chloroplast genes were tested for protein binding in a chloroplast extract. Efficient and sequence-specific RNA-protein interaction was detected with transcripts of the genes trnK, rps16 and trnH, but not with the 3' terminal region of trnQ RNA. The transacting component required for specific complex formation is probably a single 54 kDa polypeptide. The protein-binding region of the rps16 3' terminal region was mapped and compared with that of the trnK transcript determined previously. Both regions reveal a conserved 7-mer UUUAUCU followed by a stretch of U residues. Deletion of the trnK 3' U cluster resulted in more than 80% reduction in the binding activity, and after deletion of both the U stretch and the 7-mer motif no binding at all was detectable. RNase protection experiments indicate that the protein-binding regions of both the rps16 and trnK transcripts correlate with the positions of in vivo 3' ends, suggesting an essential role for the 54 kDa binding protein in RNA 3' end formation. In the case of the trnK gene, evidence was obtained for read-through transcripts that extend into the psbA coding region, thus pointing to the possibility of trnK-psbA cotranscription.
Mol Gen Genet 1991 Aug
PMID:RNA-protein interactions at transcript 3' ends and evidence for trnK-psbA cotranscription in mustard chloroplasts. 171 78

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.
J Mol Biol 1991 Sep 05
PMID:Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling. 171 95


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