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At least 19 recordsLinked to original sources
PubMed1992

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1975

Determination of mRNA half-life in HeLa cultures by a poly(A)-independent direct analysis of specific radioactivity of mRNA.

mRNA specific radioactivity in HeLa cultures exposed to (3H)uridine (10 muM) was determined directly by a highly selective poly(A)-independent method which we have described previously. Neither uridine in mRNA nor UTP approached the specific radioactivity of the exogenous (3H)uridine, but attained steady-state specific radioactivities which remained a third below the value of the added precursor. Using the labeling data for the evaluation of mRNA turnover, previously described by Greenberg, mRNA half-life in exponentially growing HeLa cultures was found to be 0.87 times the cell doubling time. Decay curves of mRNA in prelabeled cultures were in accordance with these values (half-life equals 0.79 times the cell doubling time) when corrected for growth and also for "reutilization" which was accomplished by relating uridine labeling in mRNA to UTP specific radioactivity. The experiments showed that an exact evaluation of mRNA turnover is possible only when the following points are taken into account. a) A constant supply of exogenous labeled uridine must be provided to guarantee a constant specific radioactivity of UTP. b) Labeling of CTP and of cytidine in RNA are delayed when compared with UTP and uridine in RNA. Corrections for cytidine labeling in RNA are therefore required. c) As rRNA approached a definitely lower steady-state specific radioactivity than mRNA, mRNA specific radioactivities must be determined directly (i.e. by radioactivity and absorbance at 260 nm in isolated mRNA fractions) in order to evaluate true turnover of this RNA species.

Cell Count↗
PubMed1990

In the beginning is the end: regulation of poly(A) addition and removal during early development.

The addition of poly(A) tails to nearly all mRNAs within the nucleus was reviewed in the July issue of TIBS. Here we shift focus to the fate of poly(A) tails during early development. At specific times during oogenesis and embryogenesis, the poly(A) tails of certain maternal mRNAs are lengthened, while the tails of a number of other mRNAs are removed. The selective poly(A) addition reactions are regulated by a short, U-rich sequence in the 3' untranslated region, while the removal of poly(A) from specific mRNAs is a 'default state', requiring no specific sequence. These regulated changes in poly(A) length are likely to play a major role in translational regulation in the egg and early embryo.

Animals↗
PubMed1991

Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation.

In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.

Cloning, Molecular↗
PubMed1992

Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity. Increased specificity toward src peptides.

A tyrosine protein kinase (TPK-I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella-Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123-126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c-src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 microM versus 670 microM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N-terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position -2 or glutamic acid at position -1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl. These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.

Amino Acid Sequence↗
PubMed1991

Determination of ethaverine and papaverine using ion-selective electrodes.

Ion-selective poly(vinyl chloride) membrane electrodes for the opium alkaloids papaverine and ethaverine are presented. The electrode membranes contain ion pairs of the alkaloids with the anionic counter ion tetraphenylborate. The detection limits for all electrodes were approximately 2 x 10(-6) mol dm-3 at pH 5.0 in 100 mmol dm-3 buffered solutions and the measured slopes were close to the values theoretically expected. The selectivity coefficient observed for the ethaverine-tetraphenylborate electrode is 10(-1.1) with respect to papaverine. The suitability of the membranes for single-use electrodes is discussed.

Electrochemistry↗
PubMed1991

Immunology, biosynthesis and in vivo assembly of the branched-chain 2-oxoacid dehydrogenase complex from bovine kidney.

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1 beta subunit relative to the E2 and E1 alpha polypeptides employing either purified E1 or BCOADC as antigens. Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55,000-Mr species which was identified as E3 on the basis of immunocompetition studies. Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, E1 alpha and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent Mr value of 56,500, pre-E1 alpha, 49,000 and pre-E3, 57,000 compared to subunit Mr values of 50,000, 46,000 and 55,000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence. In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1 alpha was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1977

Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance.

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.

Acid-Base Equilibrium↗
PubMed2026

Maternal PAN2 selectively maintains mRNA Poly(A) tail homeostasis to regulate RNA degradation during oocyte-to-early embryo transition in mice.

In mammals, the precise degradation of maternal mRNAs is essential for oocyte maturation and early embryonic development, as it facilitates the "maternal-to-zygotic transition (MZT)" by eliminating maternal transcripts and enabling zygotic genome activation (ZGA). However, the physiological role of the poly(A)-specific nuclease 2 (PAN2), a deadenylase that initiates cascade degradation of long-tailed transcripts, remains unknown. Here, we generated oocyte-specific Pan2 conditional knockout (cKO) mice to investigate its role. We found that Pan2 cKO females exhibit severe female subfertility despite normal oocyte maturation and ovulation, with embryos arresting at the 2-cell stage. PAIso-seq2 and transcriptome sequencing reveal that PAN2 coordinates maternal mRNA deadenylation and decay. Mechanistically, PAN2 recognizes its substrates through a PAN3-PABPC1 bridging complex, and it preferentially targets transcripts whose poly(A) tails lack guanosine (G) but are enriched for uridine (U). PAN2 deficiency causes poly(A) tail dyshomeostasis, leading to global accumulation of maternal mRNAs, impaired zygotic genome activation, and abnormal protein accumulation in 2-cell embryos. Overexpression of these proteins phenocopies developmental defects. Notably, the PAN2-regulated transcriptome is largely non-overlapping with the LC3B-mediated degradation pathway, highlighting the unique and non-redundant role of PAN2 in maternal mRNA clearance. Our study establishes maternal PAN2 as a critical regulator of poly(A) tail homeostasis, ensuring timely maternal mRNA clearance and proper ZGA, highlighting the stage-specific and tail-composition-dependent functions of the deadenylase cascade during the maternal-to-zygotic transition. These findings offer new perspectives on post-transcriptional regulatory mechanisms in early mammalian embryogenesis.

Deadenylation↗
PubMed1975

The selective action of poly A:U on the electrophoretic mobility and surface charge of a subpopulation of B cells.

Electrophoretic mobility (E.P.M.) of lymphoid cells has been determined in three species. In chicks, the E.P.M. of bursa cells was 0.47 plus or minus 0.07 and of thymocytes 0.64 plus or minus 0.10 mum/sec/volt/cm. On the basis of the E.P.M. profile of the Ficoll gradient purified lymphocytes from the rat spleen, two populations of cells present in the proportion of 65 and 35% were identifiable. The former had a mobility of 1.06 plus or minus 0.08 and the latter 1.24 plus or minus 0.09. The E.P.M. of lymphocytes prepared from athymic nude mice was 0.86 plus or minus 0.06 with a Gaussian distribution profile. On incubation of these cells with poly adenylate-poly uridylate (poly A:U) for 30 min at 38 degrees C followed by extensive washings, the E.P.M. of thymocytes from the chick and rat as well as the thymus-derived cells in rat spleen remained essentially unchanged. On the other hand, poly A:U increased the E.P.M. of avian bursa cells and of a subpopulation of B cells in rat spleen and of splenocytes from nude mice. The proportion of cells markedly affected by poly A:U was 70 to 83% of the total B cells in these species.

Animals↗
PubMed1991

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Animals↗
PubMed2025

RNA Sequencing Protocols for Short-Read Sequencing.

RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.

Gene Library↗
PubMed1991

Occlusion of the HIV poly(A) site.

To investigate the selective use of poly(A) sites in the 3' long terminal repeat (LTR) but not the 5' LTR of retroviruses, we have studied the poly(A) site of the human immunodeficiency virus (HIV-1). Using hybrid HIV/alpha-globin gene constructs, we demonstrate that the HIV poly(A) site is inactive or occluded when adjacent to an active promoter, either the homologous HIV promoter or the alpha-globin gene promoter. Furthermore, this occlusion of the HIV poly(A) site occurs over a considerable distance of up to at least 500 bp. In contrast, two nonretroviral poly(A) sites [alpha-globin and a synthetic poly(A) site] are active when close to a promoter. We also show that a short fragment of approximately 60 nucleotides containing the HIV poly(A) site is fully active when placed at the 3' end of the human alpha-globin gene or within the rabbit beta-globin gene. This result rules out the requirement of more distant upstream elements for the activity of the HIV poly(A) site, as has been suggested for other viral poly(A) sites. Finally, we show that the GT-rich downstream region of the HIV poly(A) site confers poly(A) site occlusion properties on a synthetic poly(A) site. This result focuses attention on this more variable part of a poly(A) site in retroviruses as a possible general signal for poly(A) site occlusion.

Animals↗
PubMed1977

Comparative studies on the '5'-cap' and in vitro translational activity of cytoplasmic and nuclear poly A(+) RNA1.

The translational activities of cytoplasmic poly A(+)RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15-20 times greater than tose of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles 3H-leucine incorporated/micron g cytoglasmic poly A(+) RNA for the liver and tumor respectively and 3-4 pmoles 3H-leucine incoporated/micron g nuclear poly A(+)RNA. Inasmuch as intergity of the '5'-cap' of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total 32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the '5'-cap'; in nuclear poly A(+) RNA, the '5'-cap' contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5'-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the '5'-cap'. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the '5-cap' and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the '5'-cap' was not removed during translation of the mRNA in the wheat germ system; this result suggests that the '5'-cap' may associate with allosteric binding sites of initiation factor(s).

Animals↗
PubMed1992

Liver-specific RNA processing of the ubiquitously transcribed rat fibrinogen gamma-chain gene.

Fibrinogen gamma-chains differ in amino acid sequence at the carboxyterminus due to alternative 3' RNA processing. Previous studies reported differences between humans and rats in the mechanism of gamma-chain RNA processing and that it was a nonregulated event. To test the hypothesis that rat gamma-chain RNA processing involves both alternative splicing and poly(A) site selection and that it is regulated in a tissue-specific manner, we determined the tissue distribution of gamma-chain mRNA expression and the pattern of gamma-chain pre-mRNA processing. The results of in situ hybridization demonstrated that gamma A and gamma B transcripts were localized to and codistributed in liver hepatocytes, indicating that no subset of cells process gamma B mRNA. The ubiquitous expression of the fibrinogen gamma-chain promoter was demonstrated in marrow, lung, brain, and liver by RNase protection using a 5'-specific gamma-chain probe. RNase protection studies to map 3' RNA processing sites suggested that, in addition to the distal poly(A) signal previously identified, two alternative poly(A) signals within the last intron (ATTAAA and AATAAA) were used only in liver to produce gamma B transcripts. Approximately equal usage of the three poly(A) signals (27%, 37%, and 36%, respectively) to form the 3' end of mature gamma B transcripts suggested that poly(A) site selection is random. These results indicate that splicing of the last intron to produce gamma A mRNA is the ubiquitous and constitutive pattern of gamma-chain RNA processing, while retention of the last intron to produce gamma B mRNAs is the tissue-specific and regulated pattern of gamma-chain RNA processing. The pattern of rat gamma-chain RNA processing is similar to human, implying that the mechanism is conserved. These data support a mechanism of tissue-specific splice site selection predominating over poly(A) site selection in gamma-chain pre-mRNA processing. The expression of both fibrinogen gamma-chain transcripts in liver, rather than mutually exclusive expression in liver and other tissues, provides a new model for studying tissue-specific alternative 3' end formation regulatory mechanisms.

Animals↗
PubMed1979

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.

Animals↗
PubMed1978

Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells.

The structure of the 5'-termini has been investigated in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 microgram/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA.

Base Sequence↗