Effect of Met-tRNAf deacylase on polypeptide chain initiation in rabbit reticulocyte lysate

The possible role of Met-tRNAf deacylase in the regulation of protein synthesis in rabbit reticulocyte lysate by the hemin-controlled translational repressor (HCR) or the double-stranded RNA-activated inhibitor (dsI) has been examined. Inhibition of protein synthesis by either HCR or dsI is associated with a marked increase in the steady state level of 48 S initiation complexes, containing a 40 S ribosomal subunit, globin mRNA, and a reduced level of Met-tRNAf, suggesting that the rate of 60 S subunit addition may be inhibited and that subunit-bound Met-tRNAf may become deacylated by Met-tRNAf deacylase. The addition of highly purified Met-tRNAf deacylase to lysate samples incubated with HCR or dsI reduces the [35S]Met-tRNAf labeling of 48 S complexes to even a lower level but has no effect on the high level of [35S]Met-tRNAf associated with 43 S complexes in the plus hemin control. The effect of added deacylase on the labeling of 48 S complexes with [35S]Met-tRNAf can be overcome by adding eIF-5 or a soluble reticulocyte protein that has been termed the reversing factor, but not by the addition of eIF-2. Added deacylase has no effect on the level of mRNA in 48 S complexes or the labeling of these complexes with [35S]fMet-tRNAf. When lysate samples were labeled with Met-tRNAf, purified from wheat germ or yeast, and doubly labeled with 32P at the 5' end and [35S]methionine aminoacylation, HCR reduced the level of 32P and 35S-labeled tRNAMetf in 48 S complexes to a similar degree, suggesting that once it has become deacylated, tRNAMetf dissociates from the 40 S subunit.     

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.     

Mode of inhibition of globin synthesis by m7G5'p and m7G5'ppp.

The mode of inhibition of rabbit globin synthesis by m7G5'p and m7G5'ppp ("cap analogs") was studied using the rabbit reticulocyte lysate system. The rate of globin synthesis was measured at various concentrations of both f[35S]Met-tRNAf and the cap analogs. The cap analogs were found to inhibit competitively the incorporation of f[35S]Met into hot trichloroacetic acid-insoluble material. Nascent chains prelabelled with f[35S]Met were released at various concentrations of m7G5'ppp. The release of nascent chains was not inhibited by m7G5'p (Suzuki, H. (1976) FEBS Lett. 72, 309) and m7G5'ppp, and it is therefore concluded that the cap analogs inhibit a step of initiation of globin synthesis. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of an 80S/fMet-tRNAf was inhibited by the cap analogs, but not that of a 40S/fMet-tRNAf complex. These data suggest that a factor which is required for the binding of globin mRNA with 40S/fMet-tRNAf complex forms an inactive complex with the cap analogs, so that the cap analogs inhibit globin synthesis.     

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit

Different fractions of cellular RNA from erythroid enriched bone marrow cells of the rabbit, extracted by the temperature fractionation method, were investigated by hybridization to globin cDNA. 97.4% of all globin sequences were found in the 4 degrees C franction (cytoplasmic RNA) 0.11% are in the 40 degrees / 50 degrees C fraction and 2.47% in the 65 degrees C and 85 degrees C franctions (pre-mRNA). This shows a substantial purification of the pre-mRNA fractions from cytoplasmic mRNA. 33% of the globin sequences in the 65 degrees C and 85 degrees C fractions are polyadenylated. The poly(A)+-RNA from the 65 degrees C and 85 degrees C fractions separated in a formamide sucrose gradient showed a clear hybridization to globin cDNA in the region between 9S and 28S and around 4S. In a control experiment in which RNA from baby hamster kidney cells (BHK) was mixed with globin mRNA and separated in the same manner hybridization was observed at the 9S position of the gradient only.     

The binding of Met-tRNAf to isolated 40-S ribosomal subunits and the formation of Met-tRNAf - 80-S-ribosome initiation complexes.

Different forms of 40-S ribosomal subunit, distinguishable by their buoyant densities on CsCl equilibrium density gradients, are formed when derived 40-S ribosomal subunits are incubated with partially purified reticulocyte ribosomal wash proteins. One of these subunits, the 1.37-g-cm-3 form is not present in the cell but the other two forms, the 1.40-g-cm-3 and 1.40-g-cm-3 subunits, are present in cell extracts. 35S label is bound to 1.37-g-cm-3 and 1.40-g-cm-s subunits when [35S]Met-tRANf, GTP and poly(A,U,G) are included in the incubations. The 35S-labelled 40-S subunits recovered, and the amount of 35S label bound to them, are changed if the [35S]Met-tRNAf-40-S-subunit-poly(A,U,G) complexes are first purified on sucrose gradients before analysing them on CsCl. The 1.37-g-cm-3 particle is no longer seen and the total quantity of 35S label on the 40-S subunits is 90% lower after sucrose gradient purification. Between 30% and 40% of the 40-S subunits bind [35S]Met-tRNAf when 1 mM GTP, an excess of ribosomal wash proteins and [35S]Met-tRNAf over derived 40-S subunits, and poly(A,U,G) or AUG is included in the incubations. The omission of poly(A,U,G) or AUG from the incubations substantially lowers the amount of subunit-bound 35S label ultimately recovered. With these incubations less than 10% of the 40-S subunits have bound [35S]Met-tRNAf. [35S]Met-tRNAf binding is affected by the nature of the RNA added. The addition of poly(U), rRNA and native 9-S golbin mRNA is without effect, whereas denatured globin mRNA is stimulatory. Maximum binding is obtained however with AUG. Poly(A,U,G) is less stimulatory than AUG but more stimulatory than denatured mRNA, suggesting that the number as well the accessibility of the AUG initiations condons determines the amount of 35S label bound. Similar results are obtained for the ribosomal-wash-dependent binding of [35S]Met-tRNAf to 80-S ribosomes. Contrary to the binding results, the ability of mRNA to stimulate protein synthesis is dependent on the integrity of the mRNA. Thus, native 9-S globin mRNA but not poly(A,U,G) stimulatex protein synthesis in the wheat germ system. HCHO-treated globin mRNA, although stimulatory, is 45% less effective than native mRNA. The addition of AUG, derived 60-S subunits and extra ribosomal wash is required for the formation of [35S]Met-tRNAf-80-S-ribosome complexes from sucrose-gradient-purified [35S]Met-tRNAf-40-S-subunit complexes. The 80-S ribosome complexes are able to form peptide bonds. Thus, if puromycin is added to the full incubations at zero time, no 35S label is present on the 80-S ribosome. 35S label is released as methionyl-puromycin. If the [35S]Met-tRNAf-40-S-subunit complexes are assembled with poly(A,U,G) or AUG in the incubations and then purified, only derived 60-S subunits are required to form [35S]Met-tRNAf-80-S-ribosome complexes. 35S label is not released from them when puromycin is added to the incubations unless extra ribosomal wash is also added.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

Protein synthesis in yeast Saccharomyces cerevisiae. Purification of Co-eIF-2A and 'mRNA-binding factor(s)' and studies of their roles in Met-tRNAf.40S.mRNA complex formation

Antibodies prepared against a homogeneous preparation of Co-eIF-2A20 [Ahmad et al. (1985) J. Biol. Chem. 260, 6955-6959] reacted with several polypeptides including an 80-kDa polypeptide present in a crude yeast ribosomal salt wash. This 80-kDa polypeptide, containing Co-eIF-2A (Co-eIF-2A80) activity, has been extensively purified using a two-step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co-eIF-2A20 (fraction II) and hydroxyapatite chromatography (fraction III). The factors, eIF-2 + homogeneous Co-eIF-2A80 (fraction III) promoted Met-tRNAf.40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF-2 + a partially purified Co-eIF-2A80 preparation (fraction II) promoted Met-tRNAf.40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger. This factor-promoted Met-tRNAf binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG). Other polyribonucleotide messengers tested, such as poly(U), poly(A) and poly(A,C) were completely ineffective in this binding reaction. This result indicates that the Met-tRNAf.40S.mRNA complex is formed by a direct interaction between Met-tRNAf, 40S ribosomes and the initiation site in mRNA. A mechanism has been proposed for Met-tRNAf.40S.mRNA complex formation in yeast.     

Degradation of messenger RNA, ribosomal RNA and 2-5A-dependent inhibition of protein biosynthesis

In rabbit reticulocyte lysates the addition of exogenous 2-5A leads after 10-20 minutes to the inhibition of protein synthesis. This inhibition can be blocked by rat antiserum to 2-5A. In intact ribosomes the ribosomal RNA is cleaved after 2-5A addition, but this cleavage is not in correlation with the protein synthesis shutoff. Ribosomal 5S RNA and 5,8S RNA are not cleaved even after several hours of incubation with 2-5A. The degradation of polysome associated mRNA correlates with the protein synthesis inhibition as revealed by Northern blot hybridization of globin mRNA with 32P-labelled p beta G plasmid. The addition of 2-5A antiserum to the rabbit reticulocyte lysate also inhibits the degradation of polysome bound globin mRNA.     

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.     

Characteristics of rabbit globin mRNA purification by oligo(dT) cellulose chromatography

We have purified rabbit globin mRNA using oligo(dT)-cellulose and sucrose gradient centrifugation. Both α- and β-globulin mRNA molecules behave heterogeneously with respect to their elution properties during chromatography on oligo(dT)-cellulose. Those fractions eluted at the lowest ionic strength are most active in directing cell-free globin biosynthesis. By making use of hybridization with synthetic [³H]DNA complementary to globin mRNA, we have shown that this technique can be used to quantitate the extent of mRNA purification. Thus, globin mRNA is approximately 90-fold purified from reticulocyte polysomal RNA and originally constituted slightly more than 1% of the polysomal RNA. Since more than 98% of the globin mRNA sequences are bound to oligo(dT)-cellulose, we suggest that most polysomal globin mRNAs contain a poly (A)-rich region and that this region is not of uniform length nor preponderately associated with either the α- or β-globin mRNAs. In addition, we observe that the 9S globin mRNA most resistant to dissociation from oligo (dT)-cellulose is most active in directing globin biosynthesis.     

Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle

A low molecular weight RNA species, in the 70–90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at a concentration of 20–80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)⁺mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)^-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.     

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

Biosynthesis of cat hemoglobins A and B: Amino terminal acetylation of the β chain of HbB

Acetylation of the amino terminal serine of the β chains of cat HbB occurs during synthesis of hemoglobin in a mRNA dependent rabbit reticulocyte lysate protein synthesizing system in the presence of acetyl-CoA and cat reticulocyte mRNA. Both of the major cat hemoglobins, the nonacetylated HbA and acetylated HbB, are synthesized efficiently in the rabbit lysate system. The acetylation of HbB-β chains occurs during the biosynthesis of these proteins. Radioautography of tryptic peptide maps reveals that acetylation occurs specifically at the amino terminal serine of HbB-β globin, and not on HbA-β globin or on the α chain common to both hemoglobins A and B. Because of the similarity of the structures of HbA-β and HbB-β globin, it is suggested that the amino terminal residue determines whether the peptide chain is recognized for acetylation by a ribosomal acetyltransferase.     

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.     

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,     

Use of an antibody to characterize and determine the role of the major Met-tRNAf deacylase from rabbit reticulocyte ribosomes

Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(alpha) results, secondarily, in the enzymatic deacylation of Met-tRNAf on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNAf deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNAf deacylase II and has no effect on Met-tRNAf deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNAf deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 microgram/ml poly(I X C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNAf and increase the level of Met-tRNAf bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNAf in the 48 S complexes that accumulate.