Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs

These studies were designed to determine whether small cytoplasmic RNAs and two different mRNAs (actin mRNA and histone H4 mRNA) were uniformly distributed among various subcellular compartments. The cytoplasm of HeLa S3 cells was fractionated into four RNA-containing compartments. The RNAs bound to the cytoskeleton were separated from those in the soluble cytoplasmic phase and each RNA fraction was further separated into those bound and those not bound to polyribosomes. The four cytoplasmic RNA fractions were analysed to determine which RNA species were present in each. The 7 S RNAs were found in all cytoplasmic fractions, as were the 5 S and 5.8 S ribosomal RNAs, while transfer RNA was found largely in the soluble fraction devoid of polysomes. On the other hand a group of prominent small cytoplasmic RNAs (scRNAs of 105-348 nucleotides) was isolated from the fraction devoid of polysomes but bound to the cytoskeleton. Actin mRNA was found only in polyribosomes bound to the cytoskeleton. This mRNA was released into the soluble phase by cytochalasin B treatment, suggesting a dependence upon actin filament integrity for cytoskeletal binding. A significant portion of several scRNAs was also released from the cytoskeleton by cytochalasin B treatment. Analysis of the spatial distribution of histone H4 mRNAs, however, revealed a more widely dispersed message. Although most (60%) of the H4 mRNA was associated with polyribosomes in the soluble phase, a significant amount was also recovered in both of the cytoskeleton bound fractions either associated or free of polyribosome interaction. Treatment with cytochalasin B suggested that only cytoskeleton bound, untranslated H4 mRNA was dependent upon the integrity of actin filaments for cytoskeletal binding.     

Specific mRNP complexes. Characterization of the proteins bound to histone H4 mRNAs isolated from L6 myoblasts

These studies were designed to identify the proteins associated with specific mRNAs. L6 myoblasts contain a unique poly(A)-rich H4 mRNA as well as poly(A)-minus H4 mRNA subspecies. We have characterized the proteins present in both poly(A)-rich and poly(A)-minus histone H4 mRNP complexes following ultraviolet cross-linking in vivo. In addition, the muscle-specific myosin heavy chain (MHC) mRNP complex was characterized in myoblasts. [35S]Methionine-labelled poly(A)-rich and poly(A)-minus RNP complexes were prepared from both the polysomal and free (post-polysomal) RNP compartments. From each fraction the mRNP encoding histone H4 or MHC was purified by hybrid selection to a cloned human histone H4 gene or MHC cDNA. A unique set of 6-16 proteins was found bound to each of the specific mRNP complexes. These proteins were a subset of the total population of either polysomal or free RNP proteins and some proteins appeared common among the different hybrid-selected RNP fractions. The results demonstrate that (a) mRNAs bind a different set of proteins depending upon whether they are present in the polysomal or free mRNP fraction; (b) the presence of poly(A) sequences affects the proteins which bind to H4 mRNA in the free RNP compartment.     

L-bodies are RNA–protein condensates driving RNA localization in Xenopus oocytes

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.     

Intracellular localization of messenger RNAs for cytoskeletal proteins

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.     

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.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

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).     

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.