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)     

Information relay from gene to protein: the mRNP connection

Eukaryotic messenger RNAs and their binding proteins are organized into structural units called ribonucleoprotein particles (mRNPs). Some mRNP proteins are ubiquitous, and might bind all mRNAs to ensure efficient translation. Other mRNA proteins, however, are cell-specific and bind only certain mRNAs that display regulated translation. This is particularly evident in early development, where some mRNP particles can be sequestered from the translational apparatus for months before they enter polysomes. Recent investigations suggest that these and other mRNP proteins bind specific sequences and regulate translation.     

The function of proteins that interact with mRNA

Specific proteins are associated with mRNA in the cytoplasm of eukaryotic cells. The complement of associated proteins depends upon whether the mRNA is an integral component of the polysomal complex being translated, or, alternatively, whether it is part of the non-translated free mRNP fraction. By subjecting cells to ultraviolet irradiation in vivo to cross-link proteins to mRNA, mRNP proteins have been shown to be associated with specific regions of the mRNA molecule. Examination of mRNP complexes containing a unique mRNA has suggested that not all mRNA contain the same family of associated RNA binding proteins. The function of mRNA associated proteins may include a role in providing stability for mRNA, and/or in modulating translation. With the recent demonstrations that both free and polysomal mRNPs are associated with the cytoskeletal framework, specific mRNP proteins may play a role in determining the subcellular localization of specific mRNPs.     

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.     

Small cytoplasmic RNAs from human placental free mRNPs. Structure and their effect on in vitro protein synthesis

A new family of small cytoplasmic RNA species (scRNAs) was found to be associated with human term-placental free messenger ribonucleoprotein particles (mRNPs). Placental scRNAs strongly inhibit translation of both homologous and heterologous mRNAs in a cell-free rabbit reticulocyte system. scRNAs could be resolved into at least four different RNA species. One of the RNA molecules, scRNA species 1, was the most potent protein synthesis inhibitor found among the placental scRNAs. The nucleotide sequence of the scRNA species 1 was determined. In spite of its short length, scRNA species 1 still exhibited a very strong inhibitory effect on the in vitro protein synthesis. scRNAs were found to be complexed with proteins in the form of scRNPs. Proteins of these complexes enhanced the inhibitory effect of scRNAs on in vitro translation. Experiments provided evidence that inhibition of in vitro protein synthesis by the scRNAs is not dependent upon mRNA concentration. However, inhibition can be overcome by increasing the ratio lysate/scRNAs, thus suggesting that scRNAs act on some essential component of the cell-free system. The degree of inhibition is decreased when scRNAs are added after the start of translation, suggesting that scRNAs (or scRNPs) interfere with the initiation stage of translation, probably acting on an initiation factor(s). Placental scRNAs are unique in their size, being smaller than other known scRNAs. Their association with free cytoplasmic repressed mRNPs in human placenta suggests that scRNAs play a role in the regulation of mRNP metabolism and, consequently, in the control of mRNA translation.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

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.     

Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.     

Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.     

Inhibition of protein synthesis in vesicular stomatitis virus infected Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein particle

Although host protein synthesis is preferentially inhibited, there is a steady decline in the ability of Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) to synthesize both host and viral proteins. We previously reported finding an mRNA-ribonucleoprotein particle (mRNP) that contained all five VSV mRNAs and viral N protein exclusively. This particle apparently regulates translation by sequestering a majority of the VSV mRNA made late in infection and thus rendering it unavailable for protein synthesis. In the present investigation the mRNP was also shown to inhibit in vitro protein synthesis in rabbit reticulocyte and wheat germ lysates programmed with mRNA isolated from VSV-infected cells. The synthesis of eIF-2 X GTP X Met-tRNA (ternary) complex, the first step in initiation of protein synthesis, was markedly inhibited by the mRNP. The inhibition was partially reversed by addition of purified eIF-2 to the inhibited lysate or ternary complex formation reaction. These results indicate a dual role of the mRNP in regulating protein synthesis during infection. Nucleocapsid also inhibited in vitro protein synthesis, although this inhibition was not reversed by eIF-2. Nucleocapsid did not inhibit ternary complex formation in vitro. Consequently, nucleocapsid may also regulate in vivo protein synthesis, but by a mechanism different from the mRNP.     

Cytoskeletal association of muscle-specific mRNAs in differentiating L6 rat myoblasts

The importance of the cytoskeleton in protein synthesis was studied in differentiating L6 rat myoblasts. Soluble and cytoskeletal fractions obtained after gentle, non-ionic detergent lysis of myoblasts and myotubes were analysed for the presence of ribosomes and mRNPs. Polysomal mRNPs were predominantly associated with the cytoskeletal framework and free mRNPs were present in both soluble and cytoskeletal fractions. An examination of the distribution of specific mRNAs in the polysomal and free mRNP populations of both cytoplasmic fractions revealed differences in the pattern of their distribution. It is further demonstrated that in the L6 rat myoblast system, ribosomes and mRNA (or mRNP) are not associated with the microfilaments, unlike in other systems studied.     

mRNA turnover

Nuclear RNA-binding proteins can record pre-mRNA processing events in the structure of messenger ribonucleoprotein particles (mRNPs). During initial rounds of translation, the mature mRNP structure is established and is monitored by mRNA surveillance systems. Competition for the cap structure links translation and subsequent mRNA degradation, which may also involve multiple deadenylases.     

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17–20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as old mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of -globin in all systems, while the presence of the cap analogue m⁷G(5)p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5 terminal cap. Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.     

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.     

A cytoplasmic ribonucleoprotein complex containing a small RNA inhibitor of protein synthesis

Ribonucleoprotein complexes (RNP) sedimenting between 10 and 15 S were isolated from the postpolysomal cytoplasmic fraction of embryonic chicken muscle. These RNP complexes lack mRNA but contain RNA with a sedimentation coefficient of 4.4 S. The 4.4 S RNA did not arise as a product of degradation during the course of the isolation procedure nor did it contain oligo(U)- or poly(A)-rich regions. Furthermore, the 4.4 S RNA-containing RNP complex was easily separable from free mRNPs and, therefore, is not considered as part of the free mRNP complexes. Both the 4.4 S RNA and 10 to 15 S RNP were able to inhibit translation of either "capped" or "uncapped" mRNA in a heterologous cell-free system. This inhibitory effect may result from interference of 4.4 S RNA with an early event in mRNA translation. A large number of polypeptides of Mr = 14,000 to 220,000 were present in the 10 to 15 S RNP. Among these, the most prominent polypeptides were of Mr = 36,000; 48,000; 52,000; 58,000; 65,000; 78,000; 84,000; 96,000; 105,000; 165,000; and 220,000. With the exception of the Mr = 36,000 polypeptide, these major components were also found in the nonpolysomal cytoplasmic mRNA protein complexes (free mRNP).     

Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40 S ribosomal subunits to the initiation codon of beta-globin mRNA

p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.