Translational control at the level of initiation between mRNAs for pre-existing proteins and a 22-kDa heat-shock protein of Chlamydomonas by small cytosolic RNAs

Small cytosolic RNAs (scRNAs) from human placenta inhibit translation of poly(A)-rich RNA from Chlamydomonas in the wheat germ cell-free system. The major exception is the mRNA for a nuclear-coded 22-kDa chloroplast heat-shock protein whose translation is much less affected. Evidence is presented which suggests that scRNAs do not directly interact with the mRNAs but with a factor of the wheat germ system instead. It has been found that run-off translation of polyribosomes is not impaired by scRNAs whereas the formation of initiation complexes in vitro, again with the exception of those of the mRNA for the 22-kDa heat-shock protein, is heavily affected. From this evidence we conclude that scRNAs interfere with the action of one or more of the wheat germ initiation factors and that the translation of the mRNA for the 22-kDa heat-shock protein is much less dependent upon this (these) factor(s).     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.     

Fluoride inhibition of the initiation of protein synthesis in the reticulocyte lysate cell-free system.

KF (30 mM) strongly inhibits polypeptide chain initiation in the reticulocyte lysate cell-free system.. Chain elongation is partially inhibited but proceeds to a significant extent with little initiation of new chains. Polysome breakdown is incomplete after incubations as long as 20 min. Under these conditions deacylated tRNA-Met accumulates in a fraction sedimenting faster than 120 S and thus may be associated with ribosomes bound to mRNA. Incubation of the system with KF results in the accumulation of a complex which can initiate synthesis of polypeptide chains in the presence of aurintricarboxylate; KF thus inhibits a step in initiation after that inhibited by aurintricarboxylate. The accumulation of deacylated tRNA-Met is correlated with the accumulation of the aurintricarboxylate-resistant complex. Both phenomema are dependent on KF and both are inhibited by aurintricarboxylate in the same range of concentrations which inhibit initiation of protein synthesis.     

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 capbinding 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" mRNP 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 picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. 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.     

Temperature sensitivity of protein synthesis initiation in reticulocyte cell-free systems. A ribosomal factor which protects protein synthesis inactivation at elevated temperatures.

Inactivation of protein synthesis in the reticulocyte lysate system, which occurs when the system is incubated at 42 °C, was prevented by a high concentration KCl extract of the ribosomes. The KCl extract also supported protein synthesis at 42 °C by KCl-washed ribosomes. Three factor fractions (IF.15, IF.2, and IF.25) were separated from the extract and characterized in partial reactions of initiation. The factor IF.2 could prevent the inactivation of the factor IF.15-promoted protein synthesis by the washed ribosomes at 42 °C. IF.2 also overcame the decrease in IF.15-promoted 40S subunit-Met-tRNA_f complex at 42 °C. The protective activity of IF.2 was inactivated by N-ethylmaleimide. The activities of IF.15 and IF.2 were little affected by heating the factors at 42 °C. However, prewarming of KCl-washed ribosomes at 42 °C caused decreased protein synthesis in subsequent incubation at 34 °C with unwarmed factors. These results suggest that some components other than the initiation factors may be inactivated at 42 °C, which is prevented by IF.2 in the course of protein synthesis.     

Accelerated poly(A) loss on alpha-tubulin mRNAs during protein synthesis inhibition in Chlamydomonas

Detachment of flagella in Chlamydomonas reinhardii stimulates a rapid accumulation of tubulin mRNAs. The induced tubulin mRNAs are normally rapidly degraded following flagellar regeneration, but inhibition of protein synthesis with cycloheximide prevents their degradation. alpha-Tubulin poly(A) tail lengths were measured during normal accumulation and degradation, and in cycloheximide-treated cells. To measure alpha-tubulin mRNA poly(A) chain lengths with high resolution, specific 3' fragments of alpha 1- and alpha 2-tubulin mRNAs, generated by RNase H digestion of mRNA-oligonucleotide hybrids, were sized by Northern analysis. Both alpha-tubulin mRNAs have a newly synthesized poly(A) chain of about 110 adenylate residues. The poly(A) tails shorten with time, and show an average length of 40 to 60 adenylate residues by 90 minutes after deflagellation, at which time induced alpha-tubulin mRNA is being rapidly degraded. Poly(A) loss is significantly accelerated in cycloheximide-treated cells, and this loss is not attributible simply to the longer time the stabilized molecules spend in the cytoplasm. A large fraction of alpha-tubulin mRNA accumulates as mRNA with very short poly(A) tails (less than 10 residues) in the presence of cycloheximide, indicating that deadenylated alpha-tubulin mRNAs can be stable in vivo, at least in the absence of protein synthesis. The rate and extent of poly(A) loss in cycloheximide are greater for alpha 2-tubulin mRNA than for alpha 1-tubulin mRNA. This difference cannot be attributed to differential ribosome loading. This finding is interesting in that the two mRNAs are very similar in sequence with the exception of their 3' untranslated regions.     

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.     

Translational control of the abundance of cytoplasmic poly(A) binding protein in human cytomegalovirus-infected cells

Irrespective of their effects on ongoing host protein synthesis, productive replication of the representative alphaherpesvirus herpes simplex virus type 1, the representative gammaherpesvirus Kaposi's sarcoma herpesvirus, and the representative betaherpesvirus human cytomegalovirus [HCMV] stimulates the assembly of the multisubunit, cap-binding translation factor eIF4F. However, only HCMV replication is associated with an increased abundance of eIF4F core components (eIF4E, eIF4G, eIF4A) and the eIF4F-associated factor poly(A) binding protein (PABP). Here, we demonstrate that the increase in translation factor concentration was readily detected in an asynchronous population of HCMV-infected primary human fibroblasts, abolished by prior UV inactivation of virus, and genetically dependent upon viral immediate-early genes. Strikingly, while increased mRNA steady-state levels accompanied the rise in eIF4E and eIF4G protein levels, the overall abundance of PABP mRNA, together with the half-life of the polypeptide it encodes, remained relatively unchanged by HCMV infection. Instead, HCMV-induced PABP accumulation resulted from new protein synthesis and was sensitive to the mTORC1-selective inhibitor rapamycin, which interferes with phosphorylation of the mTORC1 substrate p70 S6K and the translational repressor 4E-BP1. While virus-induced PABP accumulation did not require p70 S6K, it was inhibited by the expression of a dominant-acting 4E-BP1 variant unable to be inactivated by mTORC1. Finally, unlike the situation in alpha- or gammaherpesvirus-infected cells, where PABP is redistributed to nuclei, PABP accumulated in the cytoplasm of HCMV-infected cells. Thus, cytoplasmic PABP accumulation is translationally controlled in HCMV-infected cells via a mechanism requiring mTORC1-mediated inhibition of the cellular 4E-BP1 translational repressor.     

Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors

Light induction of light-harvesting chlorophyll a/b-binding protein (LHCP) mRNA accumulation was studied in light-dark synchronized cultures of Chlamydomonas reinhardi. LHCP mRNA accumulation was prevented by the chlorophyll-synthesis inhibitor alpha,alpha-dipyridyl which blocks late steps in the chlorophyll biosynthetic pathway and leads to the accumulation of the porphyrin intermediate magnesium protoporphyrin methyl ester. LHCP mRNA accumulated normally, however, when chlorophyll synthesis was blocked by inhibitors such as hemin and levulinic acid which interfere with early steps in the chlorophyll biosynthesis pathway prior to the formation of magnesium protoporphyrin methyl ester. Similar effects were observed in the light induction of LHCP mRNA levels in protoporphyrin IX-accumulating mutants, brc-1 and brs-1. These mutants have low levels of LHCP mRNA when grown under heterotrophic conditions in the dark where they accumulate protoporphyrin IX. However, LHCP mRNA is light-induced in brc-1 which synthesizes chlorophyll in the light and presumably consumes porphyrin intermediates in doing so. These results suggest that the chlorophyll-synthesis intermediates, magnesium protoporphyrin methyl ester and its immediate precursors, inhibit by a feedback mechanism the light induction of LHCP mRNA accumulation. Low magnesium protoporphyrin methyl ester levels permit the light-induced accumulation of LHCP mRNA, whereas high magnesium protoporphyrin methyl ester levels destabilize LHCP mRNA regardless of the illumination conditions. Preliminary experiments show that LHCP mRNA accumulation in C. reinhardi is stimulated by blue light, and not by red light which stimulates LHCP mRNA accumulation in higher plants.     

Possible involvement of poly(A) in protein synthesis.

The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect.     

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.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell-free protein synthesis system

In this work, it was discovered that the stability of mRNA in a cell-free extract could be controlled by using engineered T7 terminator sequences. Specifically, it was found that mRNA stability gradually decreased as the length of the stem structure of the T7 terminator was reduced sequentially. As a result of the controlled abundance of mRNA species, it was possible to manipulate the relative expression level of target proteins by employing the T7 terminator of adjusted stem lengths.