mRNP Particles Research Articles

Temporal Changes in the RNA Distribution between Polysomes and Postpolysomal Ribonucleoprotein Particles in Tobacco Male Gametophyte

Growth of tobacco (Nicotiana tabacum L.) pollen tube is controlled by post-transcriptionally regulated protein synthesis. Stored mRNA was found to be present in the form of messenger ribonucleoprotein particles (mRNPs) in both maturing and germinating pollen. During pollen dehydration in the anthers content of mRNPs strongly increased during dissociation of polysomes suggesting a transfer of mRNA liberated from polysomal structures into mRNP particles. Pollen germination and initial pollen tube growth characterized by rapid reassociation of ribosomes was accompanied by decrease of mRNPs indicating an involvement of mRNA of these particles in the formation of polysomes. Distribution of the particular mRNAs during the pollen activation preceeding tube growth suggests that other mechanisms along with pollen rehydration are engaged in this process and that the continuous exploitation of stored mRNPs during pollen tube growth is precisely regulated.

Identification of Mouse YB1/p50 as a Component of the FMRP-Associated mRNP Particle

Fragile X mental retardation is caused by the absence of FMRP, an RNA-binding protein found in a large mRNP complex. Although there is evidence that FMRP exists as a homo-multimer, additional proteins have been identified that associate with FMRP in the mRNP. The autosomal paralogs of FMRP, FXR1P, and FXR2P, associate with FMRP, as do nucleolin and NUFIP1, all RNA binding proteins. Using cell lines that were stably transfected with Flag-Fmr1, we identified an additional protein that coimmunoprecipitates with FMRP. The approximately 50 kDa protein was identified by mass spectrometry as mouse Y box-binding protein 1 (YB1), which is 97% identical to the core mRNP protein p50, an RNA-binding protein. An anti-p50 antiserum recognized the 50 kDa protein, confirming the identification. The association of the FMRP-mRNP with a Y box protein, the latter commonly found in mRNPs, further suggests the involvement of FMRP in translation modulation.

Immunocytochemical and Biochemical Characterization of FMRP, FXR1P, and FXR2P in the Mouse

Fragile X syndrome is caused by the absence of expression of the FMR1 gene. Both FXR1 and FXR2 are autosomal gene homologues of FMR1. The products of the three genes are belonging to a family of RNA-binding proteins, called FMRP, FXR1P, and FXR2P, respectively, and are associated with polyribosomes as cytoplasmic mRNP particles. The aim of the present study is to obtain more knowledge about the cellular function of the three proteins (Fxr proteins) and their interrelationships in vivo. We have utilized monospecific antibodies raised against each of these proteins and performed Western blotting and immunolabeling at the light-microscopic level on tissues of wild-type and Fmr1 knockout adult mice. In addition, we have performed immunoelectron microscopy on hippocampal neurons of wild-type mice to study the subcellular distribution of the Fxr proteins. A high expression was found in brain and gonads for all three proteins. Skeletal muscle tissue showed only a high expression for Fxr1p. In the brain the three proteins were colocalized in the cytoplasm of the neurons; however, in specific neurons Fxr1p was also found in the nucleolus. Immunoelectronmicrsocopy on hippocampal neurons demonstrated the majority of the three proteins in association with ribosomes and a minority in the nucleus. The colocalization of the Fxr proteins in neurons is consistent with similar cellular functions in those specific cells. The presence of the three proteins in the nucleus of hippocampal neurons suggests a nucleocytoplasmic shuttling for the Fxr proteins. In maturing and adult testis a differential expression was observed for the three proteins in the spermatogenic cells. The similarities and differences between the distribution of the Fxr proteins have implications with respect to their normal function and the pathogenesis of the fragile X syndrome.

Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P

The absence of fragile-X mental-retardation protein (FMRP) results in fragile-X syndrome. Two other fragile-X-related (FXR) proteins have been described, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA metabolism. To characterize a possible functional role of the interacting proteins the complex formation of the FXR proteins was studied in mammalian cells. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. However, Western-blotting studies on cellular homogenates containing physiological amounts of the three proteins gave different indications. Gel-filtration experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of >600 kDa, as parts of messenger ribonuclear protein (mRNP) particles associated with polyribosomes. Salt treatment shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, with molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majority of the FXR proteins do not form heteromeric complexes. Further analysis by [(35)S]methionine labelling in vivo followed by immunoprecipitation indicated that no proteins other than the FXR proteins were present in these complexes. These results suggest that the FXR proteins form homo-multimers preferentially under physiological conditions in mammalian cells, and might participate in mRNP particles with separate functions.

Sequence-independent assembly of spermatid mRNAs into messenger ribonucleoprotein particles.

During mammalian spermatogenesis, meiosis is followed by a brief period of high transcriptional activity. At this time a large amount of mRNA is stored as messenger ribonucleoprotein (mRNP) particles. All subsequent processes of sperm maturation occur in the complete absence of transcription, primarily using proteins which are newly synthesized from these stored mRNAs. By expressing transgene mRNAs in the early haploid spermatids of mice, we have investigated the sequence requirements for determining whether specific mRNAs in these cells will be stored as mRNP particles or be assembled into polysomes. The results suggest that mRNAs which are transcribed in spermatids are assembled into mRNP particles by a mechanism that acts independently of mRNA sequence. Our findings reveal a fundamental similarity between the mechanisms of translational control used in spermatogenesis and oogenesis.

Translational control in plant stress: the formation of messenger ribonucleoprotein particles (mRNPs) in response to desiccation of Tortula ruralis gametophytes

SummaryChanges in gene expression observed in vivo in response to desiccation and rehydration of the desiccation‐tolerant bryophyte Tortula ruralis are regulated by alterations in the pattern of selection of messenger RNAs, from a qualitatively constant mRNA pool, by the translational machinery. When drying rates are slow, messenger ribonucleoprotein particles (mRNPs) are formed in the drying gametophytes. A representative rehydrin mRNA, Tr288, was sequestered into these particles which were analysed using sucrose and CsCl gradients. Quantitative RT–PCR analysis of the fractions from a low salt extraction demonstrated that Tr288 mRNA migrated farther in the sucrose gradient, relative to those extractable in high salt, indicating that the transcript is associated with particles that are of higher density. RT–PCR analysis also demonstrated that the majority of Tr288 mRNA, from slowly desiccated gametophytes, is associated with particles that have buoyant densities between 1.44 and 1.64 g cm– 3 which correspond to the buoyant density range reported for mRNP particles. mRNPs that are unique to drying T. ruralis gametophytes form at least four size classes after in vivo UV cross‐linking based upon FPLC analysis. This is the first report of mRNP formation in response to a vegetative water deficit in plants.

Activities of cold-shock domain proteins in translation control.

For efficient processing, transport, storage, translation, and degradation, stretches of RNA transcripts are required in a single-stranded conformation (ssRNA). A superfamily of OB-fold proteins is characterized by preference of binding to ssRNA. This superfamily consists of proteins containing either an S1 domain (S1-D) or a cold-shock domain (CSD). In a variety of situations. S1-D or CSD proteins are found in association with DEAD-box RNA helicases and the two types of protein appear to function together to maintain regions of ssRNA. CSD proteins are commonly found bound to stored (nontranslating) mRNA, particularly during early development. Although complete removal of the CSD proteins from mRNA permits its translation in vitro, low concentrations of CSD protein on the mRNA may be required for maximal translation efficiency in vivo. Another component of stored mRNP particles in Xenopus oocytes is the protein kinase CK2, which phosphorylates the associated CSD proteins. It is argued here that the loading of CSD proteins on mRNA and the stability of the protein/mRNA complex are regulated by RNA helicase activity and protein phosphorylation.

Different targets for the fragile X-related proteins revealed by their distinct nuclear localizations.

Fragile X syndrome is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP and its structural homologues FXR1P and FXR2P form a family of RNA-binding proteins (FXR proteins). The three proteins associate with polyribosomes as cytoplasmic mRNP particles. Here we show that small amounts of FMRP, FXR1P and FXR2P shuttle between cytoplasm and nucleus. Mutant FMRP of a severely affected fragile X patient (FMRPI304N) does not associate with polyribosomes and shuttles more frequently than normal FMRP, indicating that the association with polyribosomes regulates the shuttling process. Using leptomycin B we demonstrate that transport of the FXR proteins out of the nucleus is mediated by the export receptor exportin1. Finally, inactivation of the nuclear export signal in two FXR proteins shows that FMRP shuttles between cytoplasm and nucleoplasm, while FXR2P shuttles between cytoplasm and nucleolus. Therefore, molecular dissection of the shuttling routes used by the FXR proteins suggests that they transport different RNAs.

Classification of gas5 as a multi-small-nucleolar-RNA (snoRNA) host gene and a member of the 5'-terminal oligopyrimidine gene family reveals common features of snoRNA host genes.

We have identified gas5 (growth arrest-specific transcript 5) as a non-protein-coding multiple small nucleolar RNA (snoRNA) host gene similar to UHG (U22 host gene). Encoded within the 11 introns of the mouse gas5 gene are nine (10 in human) box C/D snoRNAs predicted to function in the 2'-O-methylation of rRNA. The only regions of conservation between mouse and human gas5 genes are their snoRNAs and 5'-end sequences. Mapping the 5' end of the mouse gas5 transcript demonstrates that it possesses an oligopyrimidine tract characteristic of the 5'-terminal oligopyrimidine (5'TOP) class of genes. Arrest of cell growth or inhibition of translation by cycloheximide, pactamycin, or rapamycin-which specifically inhibits the translation of 5'TOP mRNAs-results in accumulation of the gas5 spliced RNA. Classification of gas5 as a 5'TOP gene provides an explanation for why it is a growth arrest specific transcript: while the spliced gas5 RNA is normally associated with ribosomes and rapidly degraded, during arrested cell growth it accumulates in mRNP particles, as has been reported for other 5'TOP messages. Strikingly, inspection of the 5'-end sequences of currently known snoRNA host gene transcripts reveals that they all exhibit features of the 5'TOP gene family.

Identification of a Novel mRNA-Associated Protein in Oocytes ofPleurodeles waltlandXenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the completePleurodelescDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS–PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)+RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein–protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinantPleurodelesRAP55 fragment recognize the protein fromPleurodelesandXenopus.The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.

Occurrence of Proteinaceous 10-nm Filaments throughout the Cytoplasm of Algae of the Order Dasycladales

Previously, whole-mount electron microscopy of nuclei extruded together with residual cytoplasm from the rhizoids of several algal species of the order Dasycladales has revealed the occurrence of an intra- and perinuclear network of 10-nm filaments morphologically indistinguishable from that of mammalian vimentin intermediate filaments. The present investigation demonstrates the existence of a filament system throughout the cytoplasm of the rhizoid, stalk, and apical tip of these giant cells. However, while the perinuclear 10-nm filaments interconnecting the nuclear surface with a perinuclear layer of large, electron-dense bodies filled with nucleoprotein material are of smooth appearance, those continuing within and beyond the perinuclear bodies are densely covered with differently sized, globular structures and, therefore, are of a very rough appearance. The filaments in the very apical tip of the cells are mainly of the smooth type. The transition from smooth to rough filaments seems to occur in the numerous perinuclear dense bodies surrounding the large nucleus. Digestion of the rough filaments with proteinase K removes the globules from the filament surface, revealing that throughout the nonvacuolar, intracellular space the filaments have the same basic 10-nm structure. On the other hand, gold-conjugated RNase A strongly binds to the filament-attached globules but not to the smooth, perinuclear, and the proteinase K-treated, rough filaments. In addition, an antibody raised against Xp54, a highly conserved protein which in Xenopus oocytes is an integral component of stored mRNP particles, decorates the rough but not the smooth 10-nm filaments. These results support the notion that the 10-nm filament system of Dasycladales cells plays a role in the transient storage of ribonucleoprotein particles in the cytoplasm and possibly fulfils a supportive function in the actomyosin-based transport of such material to various cytological destinations.

Messenger ribonucleoprotein complexes containing human ELAV proteins: interactions with cytoskeleton and translational apparatus.

Mammalian ELAV proteins bind to polyadenylated messenger RNAs and have specificity for AU-rich sequences. Preferred binding sites in vitro include the AUUUA pentamer and related sequences present in the 3' untranslated regions of many growth regulatory mRNAs. Human ELAV (hELAV) proteins have been implicated in post-transcriptional regulation of gene expression by their effects on the stability and translatability of growth regulatory mRNAs. We have examined the intracellular localization of ELAV proteins in neurons and in tumor cells of neuronal origin using indirect immunofluorescence, confocal microscopy and biochemical separation. Mammalian neuronal ELAV proteins are found predominantly in the cytoplasm of cells in mRNP complexes termed alpha complexes which, when associated with polysomes, form large and high density ss complexes, as assayed by glycerol and accudenz gradients, respectively. Puromycin, cytochalasin or EDTA treatments disrupt beta complexes causing the release of alpha complexes, which then appear, by confocal microscopy, as large hELAV mRNP granules associated with microtubules. Association of partially purified hELAV mRNP alpha complexes with microtubules was confirmed by in vitro reconstitution assays. Furthermore, colchicine treatment of cells suggested that association of hELAV mRNP alpha complexes with microtubules is also necessary for the formation of ss complexes. Our data suggest a model in which a subset of mRNAs is associated with microtubules as ELAV mRNP particles (alpha complexes) which, in turn, associate with polysomes to form a translational apparatus (beta complex) that is, through polysomes, associated with the microfilament cytoskeletal network. hELAV proteins in these mRNP granules may affect post-transcriptional regulation of gene expression via the intracellular transport, localization and/or translation of growth regulatory mRNAs.

FMRP Associates with Polyribosomes as an mRNP, and the I304N Mutation of Severe Fragile X Syndrome Abolishes This Association

Fragile X mental retardation is caused by the lack of FMRP, a selective RNA-binding protein associated with ribosomes. A missense mutation, I304N, has been found to result in an unusually severe phenotype. We show here that normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome.

Developmental Testis-Specific Regulation of mRNA Levels and mRNA Translational Efficiencies for TATA-Binding Protein mRNA Isoforms

Early spermatids contain roughly 1000-fold more TATA-binding protein (TBP) mRNA than do somatic cells. The appearance of TBP-overexpressing spermatids in the developing testis is accompanied by a large increase in whole-organ levels of total RNA and of poly(A)+RNA per cell. Whereas somatic cells initiate transcription of TBP mRNA at a single promoter/first exon (exon 1C), in adult testis, two additional major promoter/first exons (1D and 1E) are used. We have examined the expression of the somatic and testis-specific TBP mRNA isoforms during rodent testis development. In juvenile testes TBP mRNAs containing either exon 1C or exon 1D, but none containing exon 1E, are detected. At 21 days of age, all TBP mRNA isoforms begin to overaccumulate. The onset of TBP mRNA overaccumulation is marked first by an increase in levels of polysomal TBP mRNA, and later by accumulation of mRNP-associated TBP mRNA. In adult testes, only 30% of the total TBP mRNA is engaged by polysomes; the remainder is sequestered as mRNP particles. All of the TBP mRNA isoforms in adults exist both as free mRNP particles and as polysomes; however, the fraction in polysomes varies from 60% (exon 1C) to 10% (exon 1E). This suggests that sequences within the first exons alter the probability that the mRNA will either assemble into polysomes or into translationally inactive mRNP particles.

Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes.

In investigating the composition of stored (maternal) mRNP particles in Xenopus oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which are Y-box proteins involved in a general packaging of mRNA. We now identify a third, abundant, integral component of stored mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases. Xp54 was first detected by its ability to photocrosslink ATP. Subsequent sequence analysis identifies Xp54 as a member of a helicase subfamily which includes: human p54, encoded at a chromosomal breakpoint in the B-cell lymphoma cell line, RC-K8; Drosophila ME31B, encoded by a maternally-expressed gene, and Saccharomyces pombe Ste13, cloned by complementation of the sterility mutant ste13. Expression studies reveal that the gene encoding Xp54 is transcribed maximally at early oogenesis: no transcripts are detected in adult tissues, other than ovary. Using a monospecific antibody raised against native Xp54, its presence in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density. On isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity. Possible functions of Xp54 are discussed in relation to the assembly and utilization of mRNP particles.

The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals.

Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.

Transcription and masking of mRNA in germ cells: involvement of Y-box proteins.

Gametogenesis is directed by various specialized genetic mechanisms which, to a considerable extent, apply to the production of both eggs and sperm and have been conserved across a wide spectrum of eukaryotic organisms. Two key aspects which are discussed here are: germ-cell-specific gene transcription; and translational repression (masking) of mRNA accumulated in oocytes and spermatocytes/spermatids. Together, these two processes conspire to deliver often large amounts of essential proteins at the appropriate stages of development. It is perhaps not surprising that recent evidence points to a functional link between transcription activation and translation repression, both processes being determined in the nucleus and involving common components. One set of components which has been studied recently are members of the Y-box family of regulatory proteins. Most information of the involvement of Y-box proteins in germ cell development comes from studies on amphibian oocytes and mammalian spermatids. In these cells, Y-box proteins have been detected as major components of both maternal and paternal mRNP particles and have been shown to be instrumental in the masking process. Y-box proteins are also implicated in the regulation of several germ-cell-specific genes. Possible connections between these processes are discussed.

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20–80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.

Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes

The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.