Maternal total cell-free DNA in preeclampsia with and without intrauterine growth restriction.

Elevation of total cell-free DNA (cfDNA) in patients with preeclampsia is well-known; however, whether this change precedes the onset of symptoms remains inconclusive. Here, we conducted a nested case-control study to determine the elevation of cfDNA levels in women who subsequently developed preeclampsia. Methylated HYP2 (m-HYP2) levels were determined in 68 blood samples collected from women with hypertensive disorders of pregnancy, along with 136 control samples, using real-time quantitative PCR. The measured m-HYP2 levels were converted to multiples of the median (MoM) values for correction of maternal characteristics. The m-HYP2 levels and MoM values in patients with preeclampsia were significantly higher than in controls during the third trimester (P < 0.001, both), whereas those for women who subsequently developed preeclampsia did not differ during the second trimester. However, when patients with preeclampsia were divided based on the onset-time of preeclampsia or 10th percentile birth weight, both values were significantly higher in women who subsequently developed early-onset preeclampsia (P < 0.05, both) and preeclampsia with small-for-gestational-age (SGA) neonate (P < 0.01, both) than controls. These results suggested that total cfDNA levels could be used to predict early-onset preeclampsia or preeclampsia with SGA neonate.

Plasma levels of oxidative stress-responsive apoptosis inducing protein (ORAIP) in rats subjected to physicochemical oxidative stresses.

Oxidative stress is known to play a pivotal role in the pathogenesis of various disorders including atherosclerosis, aging and especially ischaemia/reperfusion injury. It causes cell damage that leads to apoptosis. However, the precise mechanism has been uncertain. Recently, we identified an apoptosis-inducing humoral factor in a hypoxia/reoxygenated medium of cardiac myocytes. We named this novel post-translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A) as oxidative stress-responsive apoptosis inducing protein (ORAIP). We developed a sandwich ELISA and confirmed that myocardial ischaemia/reperfusion markedly increased plasma levels of ORAIP. To investigate whether the role of ORAIP is common to various types of oxidative stress, we measured plasma ORAIP levels in rats subjected to three physicochemical models of oxidative stress including N2/O2 inhalation, cold/warm-stress (heat shock) and blood acidification. In all three models, plasma ORAIP levels significantly increased and reached a peak level at 10-30 min after stimulation, then decreased within 60 min. The (mean±S.E.M.) plasma ORAIP levels before and after (peak) stimulation were (16.4±9.6) and (55.2±34.2) ng/ml in N2/O2 inhalation, (14.1±12.4) and (34.3±14.6) ng/ml in cold/warm-stress, and (18.9±14.3) and (134.0±67.2) ng/ml in blood acidification study. These data strongly suggest that secretion of ORAIP in response to oxidative stress is universal mechanism and plays an essential role. ORAIP will be an important novel biomarker as well as a specific therapeutic target of these oxidative stress-induced cell injuries.

Platelet proteomics in thalassemia: Factors responsible for hypercoagulation.

PURPOSE: Thalassemias can be defined as a group with inherited hemolytic anemia due to differential expressions of α and ß globin genes. Hemoglobin E combined with ß thalassemia (HbEß) creates high oxidative stress in platelets producing different degrees of pathophysiological severity. Numerous cases of thalassemia have been reported with thromboembolic complications due to the hypercoagulable state, the mechanism underlying that is not yet well understood. EXPERIMENTAL DESIGN: We have used 2DE and DIGE coupled with MALDI TOF/TOF-based MS identification and characterization of altered proteins in both splenectomized and nonsplenectomized HbEß and ß thalassemia to investigate the factors responsible for hypercoagulation. RESULTS: The study revealed elevated levels of chaperones like HSP70, protein disulfide isomerase; oxidative stress proteins like peroxiredoxin2 and superoxide dismutase1 along with high ROS levels. Upregulation of translation initiation factor 5a observed in thalassemia is a novel finding and plays a protective role toward cell survival under oxidative stress. CONCLUSIONS AND CLINICAL RELEVANCE: The altered levels of chaperones and oxidative stress proteins indicate toward regulation of integrin binding and platelet activation under oxidative stress. Altogether, this comparative proteomics study of platelets in thalassemia could provide an insight into better understanding of the pathophysiology of the disease.

Prospective study of eukaryotic initiation factor 4E protein elevation and breast cancer outcome.

OBJECTIVE: To validate the authors' initial hypothesis-generating observation that eukaryotic initiation factor 4E (eIF4E) protein elevation predicts a higher cancer recurrence rate in patients with stage 1 to 3 breast cancer. SUMMARY BACKGROUND DATA: Tumor size and nodal status continue to be the two most important independent prognostic markers in breast cancer, despite well-documented limitations. In a previous smaller retrospective study, eIF4E, important in the regulation of protein synthesis of mRNAs with long or complex 5' untranslated regions, appeared promising as an independent predictor of breast cancer recurrence. METHODS: Specimens and clinical data from 191 patients with stage 1 to 3 breast cancer were accrued prospectively. Data collected include stage of disease, tumor grade, age at diagnosis, and menopausal status. Endpoints measured were disease recurrence and cancer-related death. eIF4E protein level was quantified using Western blot analysis. Immunohistochemical staining was used to determine estrogen receptor, progesterone receptor, and HER-2/neu receptor status. Statistical analysis include Cox proportional hazards model, log-rank test, Kaplan-Meier survival curve, Fisher exact test, and t test. RESULTS: Patients were divided into three groups based on tertile distribution of eIF4E: low, defined as less than 7.5-fold elevation (n = 64); intermediate, defined as 7.5- to 14-fold elevation (n = 61); and high, defined as more than 14-fold elevation (n = 66). The relative risk for cancer recurrence with intermediate elevation was 4.1 times that of patients with low elevation. For patients with high elevation, the relative risk for recurrence was higher, at 7.2 times that of the low group. The relative risk for cancer-related death for high elevation was 7.3 times that of patients with low eIF4E. Using multivariate analysis, high eIF4E remained an independent predictor of cancer recurrence after adjusting for tumor size, tumor grade, nodal disease, estrogen receptor status, progesterone receptor status, and menopausal status. CONCLUSIONS: High eIF4E is an independent predictor of cancer recurrence in patients with stage 1 to 3 breast cancer. The relative risk for cancer recurrence increases with eIF4E protein elevation. High eIF4E elevation is also associated with an increased relative risk for cancer-related death.

Eukaryotic initiation factor 5A: the molecular form of the hypusine-containing protein from human erythrocytes.

Eukaryotic initiation factor 5A (eIF-5A, formerly known as eIF-4D) purified from human erythrocytes has been found to have a monomeric molecular weight between 17,500 and 18,000. In this study, using exclusion chromatography and analytical ultracentrifugation, we demonstrate that eIF-5A normally exists as a dimer in solution and appears to be capable of undergoing reversible association to form higher polymers.

Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C.

Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.

The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase.

The rabbit reticulocyte Mr 90,000 protein associated with the heme-sensitive eIF-2 alpha kinase has been identified previously as the mammalian heat shock protein of this size class (hsp 90). Purified reticulocyte hsp 90 when added exogenously to the kinase increases its activity. This stimulatory effect is abolished after incubation of hsp 90 with a highly purified type 1 phosphoprotein phosphatase isolated from reticulocytes. Phosphorylation of dephosphorylated hsp 90 by casein kinase II but not by cAMP-dependent protein kinase restores the biological activity of hsp 90 to stimulate eIF-2 alpha phosphorylation.

Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000.

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides.

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Regulation of protein synthesis in rabbit reticulocyte lysates. Requirement of initiation factor eIF-2 holoprotein for substrate specificity of heme-regulated protein kinase.

The specificity of the heme-regulated protein kinase (HRI) was investigated further by utilizing the isolated 38,000 Da subunit (alpha subunit) polypeptide of eIF-2 as the substrate. For this purpose, the three subunit polypeptides of eIF-2 (38,000 Da, alpha; 50,000 Da, beta; and 52,000 Da, gamma) were resolved by reversed-phase high performance liquid chromatography (HPLC). Results show that HRI is incapable of phosphorylating the 38,000 Da subunit separated from the other two eIF-2 polypeptides. Data suggest that the substrate specificity of HRI is determined by the quaternary structure assumed by the alpha subunit in association with the other two subunits in the eIF-2 holoprotein.

Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue.

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits.

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).

Protein phosphorylation and translational control in reticulocytes: activation of the heme-controlled translational inhibitor by calcium ions and phospholipid.

The synthesis of globin, the major protein synthesized by reticulocytes, requires the presence of heme, the prosthetic group of hemoglobin. The absence of heme leads to the activation of a nucleotide-independent protein kinase that phosphorylates the alpha subunit of the chain initiation factor eIF-2. This modification interferes with the catalytic function of eIF-2 in protein synthesis initiation. Recent progress in our understanding of the molecular mechanism of this inhibition is briefly reviewed. The same phosphorylation is catalyzed by a different enzyme (DAI) which, while constitutive in reticulocytes, is induced by interferon in other cells. This enzyme is activated by low concentrations of double-stranded RNA in conjunction with ATP. The mechanisms of activation of these enzymes are still poorly understood. HCI is believed to form an inactive complex with heme and become active when the heme is removed by hemoglobin formation. The proinhibitor form of HCI (proHCI) is unstable in vitro and, even in the presence of heme, is irreversibly inactivated by SH-binding reagents, alkaline pH, slightly elevated temperatures, or high hydrostatic pressure. In hemin-supplemented reticulocyte lysates proHCI can also be reversibly activated by oxidized glutathione (GSSG) or NADPH depletion as well as by polyunsaturated fatty acids and by Ca2+-phospholipid. The mechanism of activation of HCI by GSSG has not been clarified although it appears to involve oxidation of proHCI SH groups to disulfides. Like activation by GSSG, the activation of HCI by polyunsaturated fatty acids and by Ca2+-phospholipid also appears to be largely due to oxidation of some of the enzyme's SH groups. There thus appear to be two fully independent mechanisms of HCI activation in reticulocyte lysates, one involving heme deficiency, the other involving oxidation of proHCI SH groups. The latter, but not the former, can be prevented or reversed by NADPH generators or dithiols. ProHCI appears to be maintained in the reduced, inactive state by a system involving NADPH, thioredoxin, and thioredoxin reductase.

The association of eIF-2 with Met-tRNAi or eIF-2B alters the specificity of eIF-2 phosphatase.

In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.

Protein synthesis in rabbit reticulocytes: requirements for Met-tRNAf . 40S preinitiation complex formation with AUG-codon and physiological mRNAs.

Under standard conditions, in the presence of GTP, highly purified eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNAf binding to 40S ribosomes. Replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP, in the above system, gave significant stimulation of Met-tRNAf binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNAf binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5'-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. This is the first report on the formation of a stable Met-tRNAf . 40S initiation complex dependent on physiological mRNAs and the factor requirements for such complex formation.

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases.

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA.

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A+) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2 alpha kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.