Binding Of Polysomes Research Articles

Abstract P3097: Post-transcriptional Mechanisms Of FTO-regulated Cardiac Contractility

Background: N6-methyladenosine (m6A), the most abundant and functionally relevant mRNA modification, is reversibly demethylated by FTO, an m6A eraser and a primary regulator of m6A in the heart. In failing hearts, decreased FTO increases m6A, critically reducing cardiomyocyte contraction. FTO overexpression improved cardiac contraction and attenuated remodeling. Here, we investigate post-transcriptional and translational mechanisms of FTO-regulation of cardiomyocyte contraction in vitro and in vivo. We hypothesize that FTO directly binds to selective cardiac contractile mRNAs at their m6A site and affects their nuclear export, polysome binding, cytoplasmic availability, and protein expression. Methods and Results: We performed immunostaining to examine the intracellular location and expression of FTO under different pathological stimuli in human ventricular cardiomyocyte cell line (AC16 cells), rat primary cardiomyocytes and mouse and human hearts. FTO was detected in both cytoplasmic and nuclear compartments, and was significantly reduced in pathological hearts compared to healthy controls. MeRIP (immunoprecipitation of m6A-RNA) sequencing analysis uncovered that FTO targets different sets of cardiac mRNAs in the cytoplasm and nucleus of cardiomyocytes. Knocking down FTO increased nuclear retention of cardiac mRNAs, which was reversed with FTO overexpression. Immunoprecipitation experiments demonstrated that FTO directly binds to cardiac mRNAs. To determine the effect of FTO on translation of select cardiac mRNAs, we isolated the polysome fraction from the cytoplasm of AC16 cells. Interestingly, our stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry analyses suggested increased expression of cardiac contractile proteins with FTO overexpression. Conclusion: We demonstrate that FTO positively regulates post-transcriptional nuclear export, polysome binding of cardiac contractile mRNAs, and regulates their translation under pathological stimuli. By combining experimental and predictive models in human and mice, we have characterized the transcriptome-proteome molecular interplay regulating cardiac function.

ABA pretreatment can alter the distribution of polysomes in salt-stressed barley sprouts

Abstract The study analyzed caryopses of barley (Hordeum vulgare) cv. Stratus. Caryopses were germinated in darkness at 20°C in three experimental setups: (a) in distilled water for 24 hours, followed by 100 mM NaCl for another 24 hours (salinity stress, SS), (b) in 100 μM of abscisic acid for the first 24 hours, followed by rinsing with distilled water to remove residual ABA, and in 100 mM NaCl for another 24 hours (ABA pretreatment + salinity stress, ABAS), (c) in distilled water only (control, C). Changes in the content of free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytomatrix-bound polysomes (CMBP) were examined in barley sprouts germinated in SS and ABAS treatments for 48 hours. In salt-stressed barley sprouts, the concentrations of membrane-bound and cytoskeleton-bound polysomes (MBP, CBP and CMBP) decreased significantly, whereas an increase was noted only in the free polysome (FP) fraction. ABA pretreatment altered the distribution of polysomes in stressed plants. The content of cytoskeletonbound polysomes (CBP and CMBP) increased, FP levels decreased, whereas no changes in MBP content were observed in response to ABA treatment. Our results suggest that plants respond to salt stress by increasing the concentrations of free polysomes that are probably released from damaged cell structures, mainly membranes. Our present and previous findings indicate that ABA could inhibit the release of FP in stressed plants by enhancing polysome binding to the cytoskeleton.

Special research presentation: bmpr signaling in the mammalian oocyte improves fertilization and influences early embryo development

Much data supports the concept that there is little to no detectable new transcription during the final stages of oocyte maturation and the initial stages of early embryo development. Therefore most of the critical events at the time of fertilization and very early embryo development rely on an orchestrated program of translation of already existing mRNAs. BMPR (Bone morphogenetic protein receptor) mRNA is increased in polysome binding during oocyte maturation. Thus, BMP signaling may be important during oocyte maturation and early embryo development, since these events are dependent on the oocyte translational program of pre-formed maternal mRNAs.

BMPR signaling in the mature mammalian oocyte affects fertilization rates and early embryo development

BMPR (Bone morphogenetic protein receptor) mRNA is increased in polysome binding during oocyte maturation, presumably reflecting increased translation of this mRNA. Therefore BMP signaling may be important during oocyte maturation and early embryo development, since these events are critically dependent on the oocyte translational program of pre-formed maternal mRNAs. We sought to assess whether activation or blockade of this receptor would affect in vitro fertilization and early embryo development.

HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion

Prolonged translation arrest in post-ischemic hippocampal CA1 pyramidal neurons precludes translation of induced stress genes and directly correlates with cell death. We evaluated the regulation of mRNAs containing adenine- and uridine-rich elements (ARE) by assessing HuR protein and hsp70 mRNA nuclear translocation, HuR polysome binding, and translation state analysis of CA1 and CA3 at 8 h of reperfusion after 10 min of global cerebral ischemia. There was no difference between CA1 and CA3 at 8 h of reperfusion in nuclear or cytoplasmic HuR protein or hsp70 mRNA, or HuR polysome association, suggesting that neither mechanism contributed to post-ischemic outcome. Translation state analysis revealed that 28 and 58 % of unique mRNAs significantly different between 8hR and NIC, in CA3 and CA1, respectively, were not polysome-bound. There was significantly greater diversity of polysome-bound mRNAs in reperfused CA3 compared to CA1, and in both regions, ARE-containing mRNAs accounted for 4-5 % of the total. These data indicate that posttranscriptional ARE-containing mRNA regulation occurs in reperfused neurons and contributes to post-ischemic outcome. Understanding the differential responses of vulnerable and resistant neurons to ischemia will contribute to the development of effective neuroprotective therapies.

Polysome-Bound Endonuclease PMR1 Is Targeted to Stress Granules via Stress-Specific Binding to TIA-1

The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional approximately 680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.

Synergistic growth inhibition by Iressa and Rapamycin is modulated by VHL mutations in renal cell carcinoma

Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFα) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel–Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation.

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.

Plant polysome binding to the actin cytoskeleton as a target for physiological regulation

One of the principal functions of the actin cytoskeleton is to serve as a support for diverse physiological and biochemical processes, protein synthesis in particular. About 25 years ago, in experiments with cultured animal cells, it was discovered that most mRNAs, ribosomes, and protein synthesis factors were attached to the actin microfilaments (Lenk et al., 1977). Later, a similar phenomenon was demonstrated for plant tissues (Davis et al., 1991; Zak et al., 1995). The physiological basis for such an attachment is evident in some cases but obscure in others. Attachment of polysomes to the microfilaments might provide for the correct spatial arrangement of the translational machinery and protein synthesis activation. If the binding of polysomes to the cytoskeleton is considered an additional level of protein synthesis regulation, its physiological control is expected but is not shown, at least for plant cells. To evaluate the proportion of the cytoskeleton-bound polysomes (CBP) in the total polysome population, we used the differential sensitivity of membranes and cytoskeleton to nonionic detergents that destroy membranes but leave microfilaments intact. A difference in the polysome peak areas in sucrose density gradients of plant tissue homogenates treated and nontreated with Triton X-100 was a rough measure for the amount of CBP (stretched areas in Fig. 2). * Corresponding author. Tel.: +7-95-432-0233; fax: +7-95-977-8018.

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii.

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.

Changes in the relative distribution and properties of smooth endoplasmic reticulum subfractions isolated from the livers of rats fed 2-acetylaminofluorene

A marked increase in the amount and relative proportion of fraction II of the smooth endoplasmic reticulum (SER II) and a concomitant decrease in the SER I fraction were observed in the liver of male Sprague—Dawley rats fed diets containing 0.05% (w/w) 2-acetylaminofluorene (AAF) for various lengths of time (3–18 weeks). The amount of rough endoplasmic reticulum (RER), which remained at control levels after 3 weeks of AAF feeding, i.e. before the appearance of hyperplastic nodules (HPN), was clearly decreased after 18 weeks of continuous AAF-feeding (HPN present). When the rats were subjected to 4 or 5 cycles of interrupted AAF feeding, similar increases in SER II were also observed both in the homogenates of HPN-containing livers, as well as in the homogenates of HPN dissected out from the surrounding liver tissue. SER II, the predominant microsomal membrane fraction in the livers of rats fed AAF for 3 weeks, showed the highest level of induction of epoxide hydrolase. A marked elevation of the manganese-dependent enhancement of polysome binding in vitro was also observed in SER II from the livers of AAF-fed rats.

53] Signal recognition particle: A ribonucleoprotein required for cotranslational translocation of proteins, isolation and properties

This chapter describes the purification of signal recognition particle (SRP), which, as a component of the translocation machinery, proves to be a valuable tool for studying the mechanism of protein translocation across the membrane of the endoplasmic reticulum. The function of SRP in the translocation process involves the recognition of the signal sequence of these proteins in their nascent state, the specific binding of polysomes synthesizing the proteins to microsomal membranes (RM) vesicles, and the initiation of the translocation event. The procedure involves salt extraction of RM, fractionation of the salt extract and hydrophobic-ion exchange chromatography, further purification of SRP, and translocation assay. The SRP is composed of six different polypeptide chains (molecular weights: 72,000, 68,000, 54,000, 19,000, 14,000, and 9000) and one molecule of RNA. Signal recognition particles reconstitute the translocation activity of K-RM, which is monitored by the cotranslational conversion of preprolactin to prolactin.

Identification of ribophorins in rough microsomal membranes from different organs of several species.

Microsomes prepared from several animal sources were analyzed for the presence of proteins corresponding to the ribophorins (I and II) which have been previously characterized in rat liver rough microsomes and appear to be involved in the binding of polysomes to endoplasmic reticulum membranes. In rough microsomal membranes from rat lacrimal gland, rabbit liver, dog and chicken pancreas, and mouse myeloma, ribophorin-like polypeptides with similar electrophoretic mobilities were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In all cases the polypeptides remained associated with sedimentable polysomes after solubilization of the microsomal membranes with nonionic detergents. Ribophorin-like polypeptides were absent from smooth microsomes. Antibodies raised against each rat liver ribophorin, purified by preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoprecipitated only the corresponding polypeptide, indicating no crossreactivity between ribophorins I and II. These antibodies also immunoprecipitated the homologous ribophorins found in microsomal preparations from other organs and species.

Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein.

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.

Thiopyronine-sensitized Photodynamic Effect on RNA Synthesis inSaccharomycesCellsin Vivo

The effect of photodynamic treatment with thiopyronine and visible light on RNA metabolism in yeast cells was investigated at different times during logarithmic growth. The results show that RNA synthesis in the nucleus of the cells is not directly inhibited photodynamically. In the endoplasmic reticulum of photodynamically treated cells one finds mRNA in about the same relative amounts and quality as in untreated control cells, but the binding of polysomes on membranes in the cytoplasm as the first step of protein synthesis is inhibited for a long time after treatment as well as the synthesis of ribosomes.

Binding of rubratoxin B to mouse hepatic microsomes and in vitro effects of the mycotoxin on polysome binding to microsomal membranes as measured by the activity of an enzyme catalyzing disulphide interchange

The mycotoxin rubratoxin B binds to some component(s) of the microsomal fraction of mouse liver. The α-β unsaturated lactone ring was a structural requirement for binding. The rubratoxin B-microsomal adduct was not separated by Sephadex G-25 filtration. The binding occurred rapidly and persisted for at least 2 hr. Sulfhydryl reagents, dithiothreitol and cysteine partially protected from and/or reversed binding. Rubratoxin B inhibited the in vitro activity of an enzyme located in the endoplasmic reticulum associated with binding of the ribosomes to the membrane.

Effect of neuraminidase, phospholipase C, and manganese ions on the ribosomal reattachment capacity of ribosome-free rough endoplasmic reticulum

The putative role of membrane-bound sialic acids in the attachment of polysomes to the membranes of the rough endoplasmic reticulum (T. Scott-Burden and A. O. Hawtrey, 1973, Biochem. Biophys. Res. Commun. 54, 1288–1295) was reinvestigated. In contrast with the previous report, it was found that although treatment of the stripped membranes with bacterial neuraminidase caused a release of 2.5 nmol of N-acetylneuraminic acid/mg of membrane protein, their capacity to reattach polysomes was not affected. Following incubation of the neuraminidase-treated membranes with labeled N-acetylneuraminic acid, nucleotide triphosphates, and an ATP-generating system, no incorporation of sialic acid into the membranes was observed. Thus, the increase in polysome binding reported by the above investigators following this treatment cannot be attributed to membrane resialylation. In all probability, the Mn 2+ present in the sialylating system was responsible for their finding. In fact, about threefold increases in the polysome-binding capacity of ribosomefree rough endoplasmic reticulum membranes were observed in the presence of 5 m m Mn 2+. Phospholipase C and Ca 2+, common contaminants of some commercial preparations of Clostridium perfringens neuraminidase, could conceivably be responsible for the effects observed by Scott-Burden and Hawtrey. This possible explanation is supported by the marked reduction of polysome-binding capacity observed after incubation of the membranes with phospholipase C from C. perfringens in the presence of Ca 2+. These studies show that neuraminidase-susceptible membrane-bound sialic acids do not play a role in the attachment of polysomes to the membranes of the rough endoplasmic reticulum and provide novel insights into this complex phenomenon.

The in vitro reassembly of rough endoplasmic reticulum: ribosome binding capacity.

Rough endoplasmic reticulum membranes were dissolved in 1% deoxycholate and the deoxycholate was then dialysed out for five days. Well-defined bilayer vesicles were formed only if the dialysis was performed at room temperature for the first six hours. The vesicles were separated into a pelleted fraction (Fraction 1) and a fluffy layer (Fraction II) by centrifugation. As measured by amino acid incorporation ability, Fraction II bound polysomes, while Fraction I did not. When smooth endoplasmic reticulum was assembled, it was found that Fraction II so derived had a polysome binding capacity which was more sensitive to increased KCl concentrations (25 mM - 100 mM KCl) and that it bound significantly more monosomes than the corresponding fraction derived from rough membranes. The SDS-polyacrylamide polypeptide patterns of the various fractions were compared.

Binding of Polysomes In Vitro with Endoplasmic Membrane Prepared from Rat Liver

Binding of Polysomes In Vitro with Endoplasmic Membrane Prepared from Rat Liver

How a single sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E 1 and E 2. This mRNA has one initiation site; core, E 1, and E 2 are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.