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PubMed2000

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.

Animals↗
PubMed2003

Mg2+-induced triplex formation of an equimolar mixture of poly(rA) and poly(rU).

Magnesium ions strongly influence the structure and biochemical activity of RNA. The interaction of Mg2+ with an equimolar mixture of poly(rA) and poly(rU) has been investigated by UV spectroscopy, isothermal titration calorimetry, ultrasound velocimetry and densimetry. Measurements in dilute aqueous solutions at 20 degrees C revealed two differ ent processes: (i) Mg2+ binding to unfolded poly(rA)*poly(rU) up to [Mg2+]/[phosphate] = 0.25; and (ii) poly(rA)*2poly(rU) triplex formation at [Mg2+]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, approximately -1.6 kcal x mol(-1) of base pairs. Volume and compressibility effects of the first process are positive, 8 cm3 x mol(-1) and 24 x 10(-4) cm3 x mol(-1) x bar(-1), respectively, and correspond to the release of water molecules from the hydration shells of Mg2+ and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 x 10(-4) cm3 x mol(-1) x bar(-1), but only a small change in volume, 1 cm3 x mol(-1). A phase diagram has been constructed from the melting experiments of poly(rA)*poly(rU) at a constant K+ concentration, 140 mM, and various amounts of Mg2+. Three discrete regions were observed, corresponding to single-, double- and triple-stranded complexes. The phase boundary corresponding to the transition between double and triple helical conformations lies near physiological salt concentrations and temperature.

Binding, Competitive↗
PubMed2006

Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails.

In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation.

Animals↗
PubMed1975

The induction of delayed hypersensitivity in guinea pigs to poly U and poly A:U.

Guinea pigs were sensitized to poly U and poly A:U so that subsequent stimulation of spleen cells from these immunized animals with poly U and poly A:U resulted in the production of migration inhibitory factor (MIF). MIF was also produced when spleen cells from animals immunized with poly A:U were cultured in the presence of mycobacterial RNA or whole viable H37 Ra cells. Negative dermal reactions were observed when guinea pigs immunized with poly A, poly A:U were skin tested wtih these same synthetic nucleotides.

Animals↗
PubMed2004

Inner-sphere complexes of divalent cations with single-stranded poly(rA) and poly(rU).

A combination of ultrasound velocimetry, density, and UV spectroscopy has been employed to study the hydration effects of binding of Mn(2+) and alkaline-earth cations to poly(rA) and poly(rU) single strands. The hydration effects, obtained from volume and compressibility measurements, are positive due to overlapping the hydration shells of interacting molecules and consequently releasing the water molecules to bulk state. The volume effects of the binding to poly(rA), calculated per mole of cations, range from 30.6 to 40.6 cm(3) mol(-1) and the compressibility effects range from 59.2 x 10(-4) to 73.6 x 10(-4) cm(3) mol(-1) bar(-1). The volume and compressibility effects for poly(rU) are approximately 17 cm(3) mol(-1) and approximately 50 x 10(-4) cm(3) mol(-1) bar(-1), respectively. The comparative analysis of the dehydration effects suggests that the divalent cations bind to the polynucleotides in inner-sphere manner. In the case of poly(rU) the dehydration effects correspond to two direct coordination, probably between adjacent phosphate groups. The optical study did not reveal any effects of cation on the secondary structure or aggregation of poly(rU). In the case of single-helical poly(rA) binding is more specific: dehydration effects correspond to three to five direct contacts and must involve atomic groups of adenines, and the divalent cations stabilize and aggregate the polynucleotide.

Adenosine↗
PubMed1992

Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.

Animals↗
PubMed1997

Visualization of poly(A)-binding protein complex formation with poly(A) RNA using atomic force microscopy.

Poly(A)-binding protein (PABP) is an RNA-binding protein that binds specifically to the poly(A) tail of messenger RNAs in eukaryotes. The PABP/poly(A) tail complex has been implicated as being important in promoting the efficient initiation of translation as well as in maintaining the integrity of the mRNA. PABP binds poly(A) cooperatively with a packing density of one PABP molecule per 25 adenosine residues. We have investigated the complexes formed between purified PABP and poly(A) RNA using atomic force microscopy (AFM). PABP alone was observed to be primarily in a monomer form with a height of 1.0 +/- 0.2 nm. Following binding to poly(A), PABP appeared to be present in variable size complexes that bound lengthwise along the RNA. This size of the PABP/poly(A) complex appeared to be maximal, suggesting that PABP binding to poly(A) may be self-limiting. Poly(A) RNA alone appeared to contain a knob-like structure that largely disappeared once PABP was bound. The use of AFM has therefore provided potential new insights into the complexes formed by this RNA-binding protein.

Aluminum Silicates↗
PubMed1979

Acute induction of joint inflammation in the rat by poly I . poly C.

Intraarticular injection of interferon inducer, double-stranded polyinosinate-polycytidylate (Poly I . C) caused acute synovitis in rats. This acute inflammatory response was accompanied by an increased concentration of prostaglandin E (PGE) in the synovial tissue. Double-stranded polyadenylate-polyuridylate (Poly A . Poly U) was less potent than Poly I . Poly C in inducing synovitis and increasing PGE concentration, while single-stranded polyinosinate (Poly I) or polycytidylate (Poly C) were inactive in these respects. Intraarticular injection of partially purified mouse fibroblast interferon also induced synovial inflammation. The present results suggest that interferon may be a mediator of viral inflammatory responses.

Animals↗
PubMed1994

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair.

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.

Animals↗
PubMed1983

Effect of hyaluronidase on cell response to the antiviral and interferon inducing activity of poly(rI) . poly(rC).

The effect of hyaluronidase on cell response to poly(rI) . poly(rC) [poly(IC)] was investigated in rabbit kidney (RK) cells. For this purpose, bovine testicular and Staphylococcal hyaluronidase preparations at various degrees of purity were used. These enzyme preparations were employed at the maximal nontoxic dose for 2 hours before poly(IC) treatment. This enzymatic pretreatment of the cells strongly inhibited the antiviral activity of poly(IC), determined by using both Herpes simplex virus type-1 and vesicular stomatitis virus. It also remarkably reduced the poly(IC)-induced interferon production. This later effect could account for the diminished antiviral activity of poly(IC) in the hyaluronidase-treated cells.

Animals↗
PubMed2001

Position and sequence requirements for poly(A) length regulation by the poly(A) limiting element.

The poly(A)-limiting element (PLE) is a cis-acting sequence that acts to limit poly(A) tail length on pre-mRNA to <20 nt. Functional PLEs are present in a number of genes, underscoring the generality of this control mechanism. The current study sought to define further the position requirements for poly(A) length regulation and the core sequence that comprises a PLE. Increasing the spacing between the PLE and the upstream 3' splice site or between the PLE and the downstream AAUAAA had no effect on poly(A) length control. However, moving the PLE from the terminal exon to either an upstream exon or intron eliminated poly(A) length control. Poly(A) length control was further evaluated using a battery of constructs in which the PLE was maintained in the terminal exon, but where upstream introns were either deleted, modified, or replaced with a polypyrimidine tract. Poly(A) length control was retained in all cases, indicating that the key feature is the presence of the PLE in the terminal exon. A battery of mutations demonstrated the importance of the 5' pyrimidine-rich portion of the element. Finally, UV crosslinking experiments identified an approximately 62-kDa protein in Hela nuclear extract that binds to a wild-type 23-nt PLE RNA oligonucleotides but not to a mutated nonfunctional form of the element.

Base Sequence↗
PubMed1988

Raman spectroscopy of Z-form poly[d(A-T)].poly[d(A-T)].

Helical structures of double-stranded poly[d(A-T)] in solution have been studied by Raman spectroscopy. While the classical right-handed conformation B-type spectra are obtained in the case of sodium chloride solutions, a Z-form Raman spectrum is observed by addition of nickel ions at high sodium concentration, conditions in which the inversion of the circular dichroic spectrum of poly[d(A-T)] is detected, similar to that observed for high-salt poly[d(G-C)] solutions [Bourtayre, P., Liquier, J., Pizzorni, L., & Taillandier, E. (1987) J. Biomol. Struct. Dyn. 5, 97-104]. The characterization of the Z-form spectrum of poly[d(A-T)] is proposed by comparison with previously obtained characteristic Raman lines of Z-form poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)] solutions and of d(CG)3 and d(CGCATGCG) crystals [Thamann, T. J., Lord, R. C., Wang, A. H.-J., & Rich, A. (1981) Nucleic Acids Res. 9, 5443-5457; Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., Rich, A., & Thomas, G. J., Jr. (1984) Nucleic Acids Res. 14, 5913-5925]. Detailed spectroscopic data are presented reflecting the reorientation of the purine-deoxyribose entities (C2'-endo/anti----C3'-endo/syn), the modification of the phosphodiester chain, and the adenosine lines in the 1300-cm-1 region. The role played by the hydrated nickel ions in the B----Z transition is discussed.

Kinetics↗
PubMed1978

Circular dichroism studies of the interaction of a limited hydrolysate of T4 gene 32 protein with T4 DNA and poly[d(A-T)].poly[d(A-T)].

gp32 I is a protein with a molecular weight of 27 000. It is obtained by limited hydrolysis of T4 gene 32 coded protein, which is one of the DNA melting proteins. gp32 I itself appears to be also a melting protein. It denatures poly[d(A-T)].poly[d(A-T)] and T4 DNA at temperatures far (50-60 degrees C) below their regular melting temperatures. Under similar conditions gp32 I will denature poly[d(A-T).poly[d(A-T)] at temperatures approximately 12 degrees C lower than those measured for the intact gp32 denaturation. For T4 DNA gp32 shows no melting behavior while gp32 I shows considerable denaturation (i.e., hyperchromicity) even at 1 degree C. In this paper the denaturation of poly[d(A-T)].poly[d(A-T)] and T4 DNA by gp32 I is studied by means of circular dichroism. It appears that gp32 I forms a complex with poly[d(A-T)]. The conformation of the polynucleotide in the complex is equal to that of one strand of the double-stranded polymer in 6 M LiCl. In the gp32 I DNA complex formed upon denaturation of T4 DNA, the single-stranded DNA molecule has the same conformation as one strand of the double-strand T4 DNA molecule in the C-DNA conformation.

Circular Dichroism↗
PubMed1988

Resonance Raman spectroscopy of complexes of the helix destabilizing proteins GP32 and GP5 with poly(rA) and poly(dA).

The bacteriophage T4 helix destabilizing protein (hdp) gp32 and its complexes with poly(rA) and poly(dA) were studied with ultra-violet resonant Raman spectroscopy. The UV-resonant Raman (UV-RR) spectrum of the complex of gp5, the coat protein of bacteriophage M13, with poly(dA) was also measured and is compared with the spectrum of the gp 32/poly(dA) complex. The excitation wavelength was 245.1 nm. This is on the far UV-side of the first absorption bands of adenine and near a "window" in the protein absorption spectrum. The overlap of fluorescence due to chromophores present in the protein and resonance Raman scattering was prevented by this choice of wavelength. The spectra of the protein/polynucleotide complexes are compared with the native nucleotide spectra measured at varying temperatures. The hyperchromicity which is expected when a nucleotide changes from a stacked to an unstacked conformation was not observed for poly(rA), neither upon temperature increase nor on protein binding. In both cases poly(dA) revealed a clear hyperchromicity. This different behavior of poly(rA) and poly(dA) is probably a consequence of their different conformations. The contributions of the proteins to the spectra is weak except for two bands, at 1550 and 1610 cm-1 due to tryptophan (in case of gp32) and one band near 1610 cm-1 due to tyrosine and phenylalanine.

Chemical Phenomena↗
PubMed1979

Polypeptides synthesized in vitro by poly(A)+ and poly(A)- mRNAs of rat ascites hepatoma AH 7974 cells.

Polysomal RNA of rat ascites hepatoma AH 7974 cells was fractionated into poly(A)+ and poly(A)- mRNAs and these RNAs were translated into polypeptides in a protein-synthesizing system derived from wheat germ. The analysis of polypeptides synthesized in vitro by two-dimensional gel electrophoresis revealed that there are three classes of polypeptides. The first group can be synthesized equally by both poly(A)+ and poly(A)- mRNAs; the second and third groups are synthesized predominantly by poly(A)+ and poly(A)- mRNAs, respectively. These results suggest that the three classes of polypeptides can be characterized by the presence or absence of poly(A) tails of the corresponding mRNAs.

Animals↗
PubMed1980

Changes in amount and synthesis of poly(A)- and poly(A)+ RNA in growing and resting cultures of Tetrahymena.

This investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo(dT) column was used to separate poly(A)+ RNA from total RNA. The rates of synthesis of poly(A)- and poly(A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly(A)+ RNA, respectively). Therefore, the relative contents of poly(A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly(A)- and poly(A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.

Animals↗
PubMed2005

Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition.

At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

3' Untranslated Regions↗
PubMed1984

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii.

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.

Chemical Phenomena↗