Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

Effects of 3′deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: Analysis of response to aldosterone

Summary Previous studies have shown that aldosterone increases transepithelial active Na⁺ transport after a latent period of about 60 min and incorporation of³H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na⁺ transport measured as short-circuit current (scc). The incorporation of³H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of³H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na⁺ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.     

The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts

Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3′ ends of mRNA. Using the two-hybrid system, we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1. Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway. These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system. The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1. Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis. Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing. These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity. Received: 6 January 1997 / Accepted: 27 February 1997     

A cell-free mRNA stability assay reveals conservation of the enzymes and mechanisms of mRNA decay between mosquito and mammalian cell lines

The rate of mRNA turnover is an important determinant of levels of gene expression. Although this process has been studied extensively in mammalian cells and yeast, relatively little is known about the mRNA decay pathways in insects. Our analysis found that the vast majority of components of the mRNA decay machinery are conserved between humans and mosquitoes. Moreover, the half-lives of Aedes albopictus mRNAs are within a similar range to those of mammalian mRNAs. In order to investigate mechanistic aspects of mRNA decay in mosquitoes, we developed an in vitro system using cytoplasmic S100 extracts from A. albopictus C6/36 cells. Using this decay assay, we show here that all the pathways of mRNA turnover that have been observed in mammalian cells (deadenylation, decapping, 3′-to-5′ exonucleolytic decay and 5′-to-3′ exonucleolytic decay) are active in C6/36 extracts. Finally, we present compelling evidence that the major deadenylase in C6/36 extracts is likely to be a homolog of the human poly(A) specific ribonuclease, PARN. Our results suggest a high level of conservation in the factors and pathways of mRNA decay between mosquitoes and humans.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Regulatory Sequences of Orthologous petD Chloroplast mRNAs are Highly Specific among Chlamydomonas Species

The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′ UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.     

Putative implication of 3′-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A putative implication 3′-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3′-terminal segment (nucleotides 1777–1811) of 18S rRNA including the last hairpin 45 was accessible for complementary interactions within 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA, when added to wheat germ cell-free protein synthesizing system, specifically inhibited translation of uncapped reporter mRNA encoding β-glucuronidase. In the 5′-untranslated region (UTR), the reporter mRNA contained a leader sequence of potato virus Y (PVY) genomic RNA with fragments complementary to the region 1777–1811. A sequence corresponding to nucleotides 291–316 of PVY, which was complementary to most of the 3′-terminal 18S rRNA segment 1777–1808, was shown to enhance translational efficiency of the reporter mRNAs when placed into 5′-UTR. The obtained results suggest that complementary interactions between 5′-UTR of mRNA and 3′-terminal segment of 18S rRNA can take place during cap-independent translation initiation.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

Hybridization of biotinylated oligo(dT) for Eukaryotic mRNA quantitation

Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using a biotinylated oligo(dT) probe, which hybridizes directly to the 3′-polyadenylated sequence of eukaryotic mRNAs. The method provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern analysis.     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage. Received: 6 June 1997 / Accepted: 5 November 1997     

An Approach to Prebiotic Synthesis of α-Oligoribonucleotides and Description of Their Properties: Selective Advantage of β-RNA over α-RNA

Oligomerization of α-adenosine 5′-phosphorimidazolide (α-ImpA) has been done in an aqueous solution using a uranyl-ion catalyst or a poly(U) template as a model process of prebiotic synthesis of RNA with α-glycosidic linkage. α-Oligoriboadenylates up to hexamer were formed from α-ImpA by the uranyl-ion catalyst. 3′-5′ Linkage was mainly formed in the oligomerization. The poly(U) template mediated the oligomerization of α-ImpA, but to a very low extent. The yield and chain length of the resulting α-oligomers were far lower than those of the corresponding β-oligomer formation under the same conditions. Physico-chemical properties of α-oligoriboadenylates are presented along with those of the corresponding β-oligoriboadenylates. The results indicate that β-RNA is more advantageous than α-RNA from the points of their synthesis and properties. Received: 10 February 1997 / Accepted: 31 March 1997     

Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3′ end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5′ UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20- to 40-fold. When the hairpin was incorporated in the 3′ UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3′ end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation.     

Investigation of the thermal stability of nucleic acids and their structural components

The thermal stability of homopolynucleotides (poly(A), poly(G), poly(C), poly(U)) and natural DNA, as well as their structural components: nucleoside (uridine), nucleotides (uridine-5′-monaphosphate, uridine-5′-diphosphate, and uridine-5′-triphosphate) and sugar (D-ribose) have been studied by the method of differential scanning microcalorimetry. The dependences of the heat flow on temperature have been obtained for the compounds having individual features in the temperature range from 20 to 400°C. All samples showed exothermic peaks at temperatures higher than 200°C (for DNA, this peak was found at a temperature of ∼160°C), which are related to processes of irreversible thermal destruction. The temperatures of thermal destruction and the effective energy of activation of this process for all compounds studied have been determined. The values of the effective heat of exothermal processes have been calculated for the polynucleotides. The experimental results indicate that there is a significant difference in the thermal stability between these homopolynucleotides and DNA, poly(G) being the most stable and DNA, the least stable. Based on the analysis of D-ribose, nucleoside, and nucleotides, it was concluded that the sugar ring is the most probable region of the destruction.     

An improved and rapid protocol for the isolation of poly(A)+-RNA from small samples of scots pine seedlings

A simple rapid method for the isolation of poly(A)⁺-RNA from pine seedlings (Pinus sylvestris) was developed. The entire procedure can be carried out in Eppendorf tubes, thus greatly minimizing the time, starting material (as little as 3–5 μg of total RNA) and the amount of chemicals required. The poly(A)⁺-RNA recovered is a suitable substrate for RT-PCR.