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.     

Microtubule-dependent distribution of mRNA in adult cardiocytes

Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.     

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.     

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)     

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.     

Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment

The deposition of proteins onto newly spliced mRNAs has far reaching consequences for their subsequent metabolism. We affinity-purified spliced human mRNPs under physiological conditions from HeLa nuclear extract and present the first comprehensive inventory of their protein composition as determined by mass spectrometry. Several proteins previously not known to be mRNP-associated were detected, including the DEAD-box helicases DDX3, DDX5, and DDX9, and the ELG, hNHN1, BCLAF1, and TRAP150 proteins. The association of some of the newly identified mRNP proteins was shown to be splicing-dependent, but not to require EJC formation. Initial recruitment of EJC proteins to the spliceosome did not require an EJC binding platform at the -20/24 region of the 5' exon. Finally, while recruitment of EJC proteins and stable EJC formation were not dependent on the cap binding complex, several of the newly identified mRNP proteins required the latter for their association with mRNPs. These results provide novel insights into the composition of spliced mRNPs and the requirements for the association of mRNP proteins with the newly spliced mRNA.     

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.     

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)     

Identification of mRNA/protein (mRNP) complexes containing Puralpha,mStaufen, fragile X protein,and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.     

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.     

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.     

An in vitro system derived from Friend erythroleukemia cells to study messenger RNA stability

A system consisting of 40-80S messenger ribonucleoprotein particles (mRNP) from stationary Friend erythroleukemia (FEL) cells was used to investigate the stability of mRNA in vitro. The majority of mRNP mRNAs were found to be stable when incubated for periods of up to ninety minutes at 37 degrees. Nonetheless, many mRNAs are greatly reduced in abundance, including ones for eucaryotic elongation factor Tu (eEF-Tu) and the 73-78 kDa polypeptide commonly found in association with the poly(A) tails of mRNA. A divalent cation dependent ribonuclease (probably an endoribonuclease) could be washed off mRNP by treatment of the particles with 0.5M NaCl. The mRNAs contained in the resultant salt washed mRNPs, including eEF-Tu, were stable when incubated in vitro.     

Characterization of mouse reticulocyte free globin mRNP

Globin messenger ribonucleoprotein particles (free and polysomal) from mouse reticulocyte lysates were characterized for their mRNA composition, translational activity as well as the proteins in direct contact with them. In contrast to the homogeneous single-peak distribution of rabbit and duck reticulocyte free mRNPs, mouse free mRNP particles were heterogeneously dispersed on the sucrose density gradient into two major domains called region I and region II. Region I appeared enriched with alpha-globin mRNP and region II with beta-globin mRNP. mRNP from both regions was translationally active. Examination of lysates prepared from beta-thalassemic mice revealed a reduction of translatable beta minor mRNP within region I, supporting the hypothesis of a compensatory recruitment of beta minor free mRNP into polysomes in beta-thalassemic mice.     

Visualization of the reconstituted FRGY2-mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.