Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.     

Poly(A)-containing messenger ribonucleoprotein complexes from sea urchin eggs and embryos: polypeptides associated with native and UV-crosslinked mRNPs

Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm3, and peak buoyant densities in Cs2SO4 in 1.42 g/cm3 after fixation with glutaraldehyde and 1.46 g/cm3 without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5-10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embryonic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

Location of RNase and RNase inhibitor on free cytoplasmic mRNA-protein particles from human placenta

Free cytoplasmic mRNPs were isolated from human placenta. An activity of RNase was associated with these particles but was mostly inhibited by a labile protein inhibitor. Both RNase and RNase inhibitor were extractable from mRNPs by 0.5 M KCl. The nature of the association of the RNase-RNase inhibitor complex with mRNPs makes it suitable as a putative system for control of expression and turnover of mRNPs and therefore of protein synthesis.Abbreviations used RNase ribonuclease - mRNPs messenger ribonucleoprotein particles - pHMB p-hydroxymercuribenzoate     

Role of cytoplasmic mRNP proteins in translation.

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per mol of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA approximately 2:1, mol/mol), this protein promotes the translation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

Poly(A)-Containing Messenger Ribonucleoprotein Complexes from Sea Urchin Eggs and Embryos: Polypeptides Associated with Native and UV-Crosslinked mRNPs

Abstract. Fertilization of sea urchin eggs results in the rapid recruitment of stored messages into polyribosomes. Whether translational control in sea urchin eggs is mediated by macromolecules associated with the stored messages remains unknown, since preparations of messenger ribonucleoprotein complexes (mRNPs) were active in protein synthesis in a rabbit reticulocyte lysate. To facilitate the study of mRNPs, chromatography on oligo(dT)-cellulose was used to purify poly(A)-containing mRNPs from eggs and embryos of the sea urchin Strongylocentrotus purpuratus . Nonpolyribosomal mRNPs purified from eggs had a similar sedimentation in sucrose to unpurified mRNPs, a peak buoyant density in metrizamide of 1.22 g/cm³, and peak buoyant densities in Cs₂SO₄ in 1.42 g/cm³ after fixation with glutaraldehyde and 1.46 g/cm³ without fixation. Nonpolyribosomal mRNPs from eggs and zygotes contained 5–10 major proteins on sodium dodecylsulfate (SDS) polyacrylamide gels, and numerous minor bands. UV-irradiation of living eggs of the sea urchin Arbacia punctulata produced cross-linked mRNPs which contained a similar pattern of polypeptides to noncross-linked mRNPs. The polypeptides associated with embyronic polyribosomal mRNPs were also qualitatively similar to those present in nonpolyribosomal mRNPs, although stoichiometric differences may exist.     

A special repressor/activator system controls distribution of mRNA between translationally active and inactive mRNPs in rabbit reticulocytes

Translation of free mRNPs and polyribosomal mRNPs from rabbit reticulocytes was studied in a rabbit reticulocyte and wheat germ cell-free systems. It has been shown that translation efficiency of polyribosomal mRNPs and the mRNA isolated from the particles is nearly the same in both systems. At the same time, mRNP's translatability, which is high in the homologous cell-free system, is very low in the system from wheat germs. Translation efficiency of free mRNPs in the wheat germ system can be restored by addition of 0.5 M K CI-wash of rabbit reticulocyte ribosomes. The results testify to the existence of some special repressor repressor/activator system which controls the distribution of mRNA between free mRNPs and polyribosomes in rabbit reticulocytes.     

Translational active mRNPs from rabbit reticulocytes are qualitatively different from free mRNA in their translatability in cell-free system

The translatability of polyribosomal and free mRNPs from rabbit reticulocytes and their mRNA was compared. Both classes of mRNPs turned out to be active in rabbit reticulocyte lysates. Considerable differences between mRNPs and mRNA have been revealed. The most striking feature of mRNPs was that high concentrations of mRNPs do not inhibit protein biosynthesis, whereas high concentrations of mRNA strongly inhibit this process. This inhibition is specific for mRNA and does not occur at the addition of the same amount of rRNA from E. coli. The features of mRNP translation are not the result of addition of the supplementary translation factors within particles. The specific function of mRNP proteins in the process of translation is under discussion.     

Plant stress granules and mRNA processing bodies are distinct from heat stress granules

Similar to the situation in mammalian cells and yeast, messenger ribonucleo protein (mRNP) homeostasis in plant cells depends on rapid transitions between three functional states, i.e. translated mRNPs in polysomes, stored mRNPs and mRNPs under degradation. Studies in mammalian cells showed that whenever the dynamic exchange of the components between these states is disrupted, stalled mRNPs accumulate in cytoplasmic aggregates, such as stress granules (SGs) or processing bodies (PBs). We identified PBs and SGs in plant cells by detection of DCP1, DCP2 and XRN4, as marker proteins for the 5'-->3' mRNA degradation pathway, and eIF4E, as well as the RNA binding proteins RBP47 and UBP1, as marker proteins for stored mRNPs in SGs. Cycloheximide-inhibited translation, stress treatments and mutants defective in mRNP homeostasis were used to study the dynamic transitions of mRNPs between SGs and PBs. SGs and PBs can be clearly discriminated from the previously described heat stress granules (HSGs), which evidently do not contain mRNPs. Thus, the role of HSGs as putative mRNP storage sites must be revised.     

Alkaline phosphatase and protein kinase(s) activities in free cytoplasmic mRNPs from human term placenta

Free mRNPs isolated from human term placental tissue were examined for protein kinase and phosphoprotein-phosphatase activities. Free mRNPs incubated with [-³²P]ATP in a protein kinase standard buffer show self-phosphorylation in the absence of exogenous substrates. Treatment of phosphorylated products with alkali showed a significant phosphorylation of tyrosine residues within the mRNP-proteins. An alkaline-phosphatase activity was found to be tightly associated with the mRNPs. Both heat stable and heat labile alkaline phosphatase activities were found in the mRNPs. Heat labile alkaline phosphatase is the major isoenzyme form of the mRNPs. The existence of both protein kinase(s) and alkaline phosphatase activities in placental free cytoplasmic mRNPs might suggest that a balance between phosphorylation, specifically on tyrosine residues, and dephosphorylation states of some of the mRNP-proteins is relevant for their physiological functions, and may therefore play a role in the regulation of mRNPs' metabolism and, consequently, in mRNA translation.     

Molecular Composition of Staufen2-Containing Ribonucleoproteins in Embryonic Rat Brain

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Numerous mRNA-binding and regulatory proteins within mRNPs finely regulate the fate of bound-mRNAs. Their specific combination defines different types of mRNPs that in turn are related to specific synaptic functions. One of these mRNA-binding proteins, Staufen2 (Stau2), was shown to transport dendritic mRNAs along microtubules. Its knockdown expression in neurons was shown to change spine morphology and synaptic functions. To further understand the molecular mechanisms by which Stau2 modulates synaptic function in neurons, it is important to identify and characterize protein co-factors that regulate the fate of Stau2-containing mRNPs. To this end, a proteomic approach was used to identify co-immunoprecipitated proteins in Staufen2-containing mRNPs isolated from embryonic rat brains. The proteomic approach identified mRNA-binding proteins (PABPC1, hnRNP H1, YB1 and hsc70), proteins of the cytoskeleton (α- and β-tubulin) and RUFY3 a poorly characterized protein. While PABPC1 and YB1 associate with Stau2-containing mRNPs through RNAs, hsc70 is directly bound to Stau2 and this interaction is regulated by ATP. PABPC1 and YB1 proteins formed puncta in dendrites of embryonic rat hippocampal neurons. However, they poorly co-localized with Stau2 in the large dendritic complexes suggesting that they are rather components of Stau2-containing mRNA particles. All together, these results represent a further step in the characterization of Stau2-containing mRNPs in neurons and provide new tools to study and understand how Stau2-containing mRNPs are transported, translationally silenced during transport and/or locally expressed according to cell needs.     

Storage of messenger RNA in eukaryotes: Envelopment with protein,translational barrier at 5′ side,or conformational masking by 3′ side?

Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA-binding protein forming mRNPs and belonging to a special p50 family of basic, glycine-rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA-specific repressor proteins bound to the 5′-untranslated regions (5′-UTR) of mRNAs may be involved. Interactions of the 3′-untranslated regions (3′-UTR) with sequence-specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3′-UTR–induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible. © 1994 Wiley-Liss, Inc.     

Polysome-associated proteins in herpes simplex virus-infected cells

In the cytoplasm of eucaryotic cells, mRNA is associated with proteins. These mRNA-protein complexes, termed messenger ribonucleoprotein (mRNP) particles, are divided into two functional classes. The first class contains free (non-ribosome-associated) mRNPs which have been termed informosomes by others. The second class of mRNPs, those associated with polysomes, are actively engaged in protein synthesis and are termed polysomal mRNPs. The experiments described in this paper examined the proteins associated with polyribosomes in uninfected and herpes simplex virus type 1-infected cells. The data indicate that after infection with herpes simplex virus type 1, specific changes occur in the proteins which normally are found associated with these polysomal mRNPs. These changes include both the appearance of new and possibly virus-specific proteins and the loss of normal host-specific proteins. The relationship of these changes to the patterns of protein synthesis in these cells is also discussed.     

Assembling an intermediate filament network by dynamic cotranslation

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these "translation factories." This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation.     

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.