mRNP Particles Research Articles

Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations or deletions in the survival motoneuron 1 (SMN1) gene, resulting in deficiency of the SMN protein that is essential for motoneuron function. Smn depletion in mice disturbs axonal RNA transport and translation, thereby contributing to axon growth impairment, muscle denervation, and motoneuron degeneration. However, the mechanisms whereby Smn loss causes axonal defects remain unclear. RNA localization and translation in axons are controlled by RNA-binding proteins (RBP) and we recently observed that the neuronal RBP Ptbp2 modulates axon growth in motoneurons. Here, we identify Smn as an interactor of Ptbp2 in the cytosolic compartments of motoneurons. We show that the expression level of Ptbp2 is reduced in axons but not in the somata of Smn-depleted motoneurons. This is accompanied by reduced synthesis of the RBP hnRNP R in axons. Re-expression of Ptbp2 in axons compensates for the deficiency of Smn and rescues the defects in axon elongation and growth cone maturation observed in Smn-deficient motoneurons. Our data suggest that Ptbp2 and Smn are components of cytosolic mRNP particles, contributing to the precise spatial and temporal control of protein synthesis within axons and axon terminals.

Interplay between TPR nucleoporin and TREX‐2 complex in mRNA export

Nuclear pore complexes (NPCs) are conduits between the cytoplasm and the nucleus. NPCs consist of roughly 35 distinct nucleoporins with filaments extending from both the nuclear and cytoplasmic faces of the pore. Nucleoporins regulate many processes, including nucleocytoplasmic trafficking, protein modification, chromatin remodeling and transcription, as well as mRNA processing and export. Our current research is focused on basket nucleoporin TPR, which works as a docking platform for other proteins, including transport receptors and subunits of the TRanscription and EXport 2 (TREX‐2) complex. We found that Auxin Inducible Degron (AID)‐mediated TPR elimination specifically disrupts TREX‐2 complex recruitment to NPCs, and results in rapid and profound changes in transcriptomic profiles of TREX‐2‐dependent RNAs and retention of those RNAs within the nucleus. The molecular mechanism through which TPR controls TREX‐2‐dependent gene expression is not clear.We are currently working in two ways to understand TREX‐2‐dependent RNA export and how TPR facilitates this pathway. First, we are examining the components of TREX‐2. GANP is a major scaffold subunit of the TREX‐2 complex, and loss of GANP disrupts the association of TREX‐2 complex subunits to the NPC. We have AID‐tagged other TREX‐2 complex members (PCID2, ENY2, CETN2) and are analyzing how their loss impacts TREX‐2 targeting, transcriptomic profiles and RNA retention. Our findings suggest that GANP or PCID2 depletion rapidly leads to polyA RNA retention inside the nucleus, but this was not the case after depletion of ENY2 or CETN2. Further comparison of TREX‐2 complex subunit profiles to each other should elucidate the sequence of their actions during mRNP maturation.Second, we are investigating the point(s) at which TPR controls TREX‐2‐dependent gene expression: TPR may help to remodel mRNP particles within the nucleus, to recruit ready‐to‐export mRNP particles to the nuclear envelope, or it may perform both of these functions. To distinguish between these possibilities, we have analyzed a TPR mutant that lacks an NPC recruitment domain, to see whether it can rescue any part of TPR’s function in this pathway without recruiting TREX‐2 to the NPC. We found that this TPR mutant resides inside the nucleus and can not restore normal export of polyA RNA. We speculate that TPR preferentially acts as a landing platform for TREX‐2‐bound mRNAs at the nuclear pore and may help with quality control of the transcripts. To explore if TPR‐dependent transcripts have unique signatures, we are analyzing mRNA length, GC content and intron context in other AID‐tagged nucleoporins and comparing transcriptomic profiles after their loss in comparison to TPR.Alltogether, these data will help us to unfold the role of TREX2 complex subunits in targeting GANP‐TPR dependent transcripts to the pore and to gain a better understanding of how TPR controls gene expression.

MRNP Transport in Eukaryots: Transport of mRNP Particles in Cytoplasm

After synthesis and processing in the nucleus, mature mRNA penetrates through nuclear pores to cytoplasm. mRNP particles are formed in the nucleus and remodeled in cytoplasm, resulting in association with factors required for mRNP proper functions. In the cytoplasmic compartment, mRNP particles can include proteins participating in nuclear interactions, as well as new factors specific for cytoplasmic mRNA–protein interactions. Functions of newly synthesized mRNAs and their cytoplasmic localization are substantially diverse. Cytoskeletal and motor proteins were shown to participate in delivery of mRNP particles to their localization sites. Several families of motor proteins are known in eukaryotes, and functions of all of them were shown to be coordinated in order to compose machinery for cargo delivery to specific cytoplasmic destinations. Adapter proteins, mediators between mRNP particles and motor proteins, are also required to make this transport system functional. Unlike the well-studied system of the cytoskeletal and motor proteins, our knowledge of how mRNP particles bind motor proteins via adapter ones is still sparse.

MRNP Transport in Eukaryotes. mRNP Export from the Nucleus

Newly synthesized mRNA undergoes several stages of maturation, and the proteins involved in these processes form the mRNP particle. Mature mRNA passes through the nuclear pore from the nucleus to the cytoplasm, where it is transported to the ribosome and mRNA is translated into protein. Recruiting the export and transport factors to the newly synthesized mRNA occurs partly cotranscriptionally. Many of the proteins associate with mRNA from the earliest stages of transcription to its localization in the cytoplasm. Other factors perform their functions strictly at a certain stage. The protein composition of the mRNP particle changes during its adherence to the nuclear pore, passing through it, and being delivered to the site of localization in the cytoplasm. This paper describes the main participants in the export of the mRNP particle, starting with its delivery to the nuclear pore and its attachment to and translocation through the nuclear pore.

Neuronal messenger ribonucleoprotein transport follows an aging L\xe9vy walk

Localization of messenger ribonucleoproteins (mRNPs) plays an essential role in the regulation of gene expression for long-term memory formation and neuronal development. Knowledge concerning the nature of neuronal mRNP transport is thus crucial for understanding how mRNPs are delivered to their target synapses. Here, we report experimental and theoretical evidence that the active transport dynamics of neuronal mRNPs, which is distinct from the previously reported motor-driven transport, follows an aging Lévy walk. Such nonergodic, transient superdiffusion occurs because of two competing dynamic phases: the motor-involved ballistic run and static localization of mRNPs. Our proposed Lévy walk model reproduces the experimentally extracted key dynamic characteristics of mRNPs with quantitative accuracy. Moreover, the aging status of mRNP particles in an experiment is inferred from the model. This study provides a predictive theoretical model for neuronal mRNP transport and offers insight into the active target search mechanism of mRNP particles in vivo.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionallyidentical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than ondistal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side ofthe NPC.

Interactions of the TREX-2 complex with mRNP particle of β-tubulin 56D gene

Interactions of the TREX-2 complex with mRNP particle of β-tubulin 56D gene

A continuum of mRNP complexes in embryonic microRNA-mediated silencing.

MicroRNAs (miRNAs) impinge on the translation and stability of their target mRNAs, and play key roles in development, homeostasis and disease. The gene regulation mechanisms they instigate are largely mediated through the CCR4–NOT deadenylase complex, but the molecular events that occur on target mRNAs are poorly resolved. We observed a broad convergence of interactions of germ granule and P body mRNP components on AIN-1/GW182 and NTL-1/CNOT1 in Caenorhabditis elegans embryos. We show that the miRISC progressively matures on the target mRNA from a scanning form into an effector mRNP particle by sequentially recruiting the CCR4–NOT complex, decapping and decay, or germ granule proteins. Finally, we implicate intrinsically disordered proteins, key components in mRNP architectures, in the embryonic function of lsy-6 miRNA. Our findings define dynamic steps of effector mRNP assembly in miRNA-mediated silencing, and identify a functional continuum between germ granules and P bodies in the C. elegans embryo.

Interactions of the TREX-2 complex with mRNP particle of β-tubulin 56D gene

mRNA transport from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. A pre-mRNA molecule undergoes modification, such as 5'-capping, splicing, and 3'-end processing, in the nucleus. The molecule being modified interacts with a large number of proteins and, thus, mRNP particles are formed. The binding of factors involved in nuclear export also occurs during transcription and mRNA processing. We have shown that the functioning of TREX-2, an mRNA export complex, is restricted to the nucleus. We used the method of RNA coprecipitation that enables the selective extraction of RNA-protein complexes from samples to show that the transcription elongation complex TREX interacts with mRNA of the β-tubulin 56D gene over the entire length of the molecule. The capping protein Cbp80 reacted both with the cap structure and with a specific part of the coding mRNA of the β-tubulin 56D gene. The TREX-2 complex that mediates mRNA export from the nucleus to the cytoplasm is bound to the same part of the coding sequence. Thus, we identified a common binding site for all of the complexes under investigation on the mRNA of β-tubulin 56D. Co-immunoprecipitation reactions performed with S2 cell extracts revealed interactions between the components of complexes involved in transcription elongation, maturation, and export of mRNA. The model of molecular folding for the mRNP particle involving the mRNA of β-tubulin 56D has been proposed.

Morphogens Reveal the Appearance and Functions of lncRNAs

During the first meiotic division, the entire genetic information from DNA is transcribed into mRNPs and stored in the ovoplasm in the form of mRNP particles. The 39 human nuclear HOX proteins bind to thousands of mRNAs transcribed repeatedly by lampbrush chromosomes. HOX proteins suppress processing and translation. The RNP particles containing lncRNAs+HOX proteins are the morphogens ("transcription factors," more precisely differentiation factors), which unlock new genes and differentiate the cells of the developing embryo. All ovoplasmic mRNAs bound with HOX proteins do not translate and are noncoding. Their destination (purpose) is transportation of HOX proteins to the complementary DNAs and cell differentiation.

The human TREX-2 complex is stably associated with the nuclear pore basket

In eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC. Here, we characterize the dynamics of human TREX-2 and show that it stably associates with the NPC basket. Moreover, the association of TREX-2 with the NPC requires the basket nucleoporins NUP153 and TPR, but is independent of transcription. Differential profiles of mRNA nuclear accumulation reveal that TREX-2 functions similarly to basket nucleoporins, but differently from NXF1. Thus, our results show that TREX-2 is an NPC-associated complex in mammalian cells and suggest that it is involved in putative NPC basket-related functions.

P-Bodies and Stress Granules: Possible Roles in the Control of Translation and mRNA Degradation

The control of translation and mRNA degradation is important in the regulation of eukaryotic gene expression. In general, translation and steps in the major pathway of mRNA decay are in competition with each other. mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. Analyses of P-bodies and stress granules suggest a dynamic process, referred to as the mRNA Cycle, wherein mRNPs can move between polysomes, P-bodies and stress granules although the functional roles of mRNP assembly into higher order structures remain poorly understood. In this article, we review what is known about the coupling of translation and mRNA degradation, the properties of P-bodies and stress granules, and how assembly of mRNPs into larger structures might influence cellular function.

Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy

Nuclear export of mRNA is a key transport process in eukaryotic cells. To investigate it, we labeled native mRNP particles in living Chironomus tentans salivary gland cells with fluorescent hrp36, the hnRNP A1 homolog, and the nuclear envelope by fluorescent NTF2. Using light sheet microscopy, we traced single native mRNA particles across the nuclear envelope. The particles were observed to often probe nuclear pore complexes (NPC) at their nuclear face, and in only 25% of the cases yielded actual export. The complete export process took between 65 ms up to several seconds. A rate-limiting step was observed, which could be assigned to the nuclear basket of the pore and might correspond to a repositioning and unfolding of mRNPs before the actual translocation. Analysis of single fluorescent Dbp5 molecules, the RNA helicase essential for mRNA export, revealed that Dbp5 most often approached the cytoplasmic face of the NPC, and exhibited a binding duration of approximately 55 ms. Our results have allowed a refinement of the current models for mRNA export.

DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation

Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation1,2,3. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle which is its functional form and determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles. This led to the identification of Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD as subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.

Drosophila glutamate receptor mRNA expression and mRNP particles

The processes controlling glutamate receptor expression early in synaptogenesis are poorly understood. Here, we examine glutamate receptor (GluR) subunit mRNA expression and localization in Drosophila embryonic/larval neuromuscular junctions (NMJs). We show that postsynaptic GluR subunit gene expression is triggered by contact from the presynaptic nerve, approximately halfway through embryogenesis. After contact, GluRIIA and GluRIIB mRNA abundance rises quickly approximately 20-fold, then falls within a few hours back to very low levels. Protein abundance, however, gradually increases throughout development. At the same time that mRNA levels decrease following their initial spike, GluRIIA, GluRIIB, and GluRIIC subunit mRNA aggregates become visible in the cytoplasm of postsynaptic muscle cells. These mRNA aggregates do not colocalize with eIF4E, but nevertheless presumably represent mRNP particles of unknown function. Multiplex FISH shows that different GluR subunit mRNAs are found in different mRNPs. GluRIIC mRNPs are most common, followed by GluRIIA and then GluRIIB mRNPs. GluR mRNP density is not increased near NMJs, for any subunit; if anything, GluR mRNP density is highest away from NMJs and near nuclei. These results reveal some of the earliest events in postsynaptic development and provide a foundation for future studies of GluR mRNA biology.

The Me31B DEAD-Box Helicase Localizes to Postsynaptic Foci and Regulates Expression of a CaMKII Reporter mRNA in Dendrites of Drosophila Olfactory Projection Neurons

mRNP granules at adult central synapses are postulated to regulate local mRNA translation and synapse plasticity. However, they are very poorly characterized in vivo. Here, in Drosophila olfactory synapses, we present early observations and characterization of candidate synaptic mRNP particles, one of which contains a widely conserved, DEAD-box helicase, Me31B. In Drosophila, Me31B is required for translational repression of maternal and miRNA-target mRNAs. A role in neuronal translational control is primarily suggested by Me31B's localization, in cultured primary neurons, to neuritic mRNP granules that contain: (i) various translational regulators; (ii) CaMKII mRNA; and (iii) several P-body markers including the mRNA hydrolases, Dcp1, and Pcm/Xrn-1. In adult neurons, Me31B localizes to P-body like cytoplasmic foci/particles in neuronal soma. In addition it is present to synaptic foci that may lack RNA degradative enzymes and localize predominantly to dendritic elements of olfactory sensory and projection neurons (PNs). MARCM clones of PNs mutant for Me31B show loss of both Me31B and Dcp1-positive dendritic puncta, suggesting potential interactions between these granule types. In PNs, expression of validated hairpin-RNAi constructs against Me31B causes visible knockdown of endogenous protein, as assessed by the brightness and number of Me31B puncta. Knockdown of Me31B also causes a substantial elevation in observed levels of a translational reporter of CaMKII, a postsynaptic protein whose mRNA has been shown to be localized to PN dendrites and to be translationally regulated, at least in part through the miRNA pathway. Thus, neuronal Me31B is present in dendritic particles in vivo and is required for repression of a translationally regulated synaptic mRNA.

Xtr, a plural tudor domain‐containing protein, coexists with FRGY2 both in cytoplasmic mRNP particle and germ plasm in Xenopus embryo: Its possible role in translational regulation of maternal mRNAs

Xtr is present exclusively in early embryonic and germline cells. We have previously shown that loss-of-function of the Xtr in embryos causes arrest of karyokinesis progression. Since Xtr contains plural tudor domains, which are known to associate with target proteins directly, we examined Xtr-interacting proteins by immunoprecipitation with an anti-Xtr monoclonal antibody and detected a few RNA-binding proteins such as FRGY2, a component of messenger ribonucleoprotein (mRNP) particle. The coexistence of Xtr with FRGY2 by constituting an mRNP particle was further confirmed by gel filtration assay. Search of mRNAs in the immunoprecipitate with Xtr suggested that the Xtr-associated molecules included several mRNAs, of which translational products were known to play crucial roles in karyokinesis progression (RCC1, XRHAMM, and so on) and in germ cell development (XDead end). Immunohistochemical observation clearly showed the co-localization of Xtr with FRGY2 also in germ plasm, in which XDead end mRNA has been shown to be localized specifically. Taken together, we proposed the possible role of Xtr in translational activation of the maternal mRNAs repressed in mRNP particle.

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

Messenger ribonucleoprotein particles (mRNPs) move randomly within nucleoplasm before they exit from the nucleus. To further understand mRNP trafficking, we have studied the intranuclear movement of a specific mRNP, the BR2 mRNP, in salivary gland cells in Chironomus tentans. Their polytene nuclei harbor giant chromosomes separated by vast regions of nucleoplasm, which allows us to study mRNP mobility without interference of chromatin. The particles were fluorescently labeled with microinjected oligonucleotides (DNA or RNA) complementary to BR2 mRNA or with the RNA-binding protein hrp36, the C. tentans homologue of hnRNP A1. Using high-speed laser microscopy, we followed the intranuclear trajectories of single mRNPs and characterized their motion within the nucleoplasm. The Balbiani ring (BR) mRNPs moved randomly, but unexpectedly, in a discontinuous manner. When mobile, they diffused with a diffusion coefficient corresponding to their size. Between mobile phases, the mRNPs were slowed down 10- to 250-fold but were never completely immobile. Earlier electron microscopy work has indicated that BR particles can attach to thin nonchromatin fibers, which are sometimes connected to discrete fibrogranular clusters. We propose that the observed discontinuous movement reflects transient interactions between freely diffusing BR particles and these submicroscopic structures.

RAP55: Insights into an evolutionarily conserved protein family

The RAP55 protein family is evolutionarily conserved in eukaryotes. Two highly conserved paralogues, RAP55A and RAP55B, exist in vertebrates; their functional properties and expression patterns remain to be compared. RAP55 proteins share multiple domains: the LSm14 domain, a serine/threonine rich region, an FDF (phenylalanine–aspartate–phenylalanine) motif, an FFD-TFG box and RGG (arginine–glycine–glycine) repeats. Together these domains are responsible for RAP55 proteins participating in translational repression, incorporation into mRNP particles, protein–protein interactions, P-body formation and stress granule localisation. All RAP55A proteins localise to P-body-like complexes either in the germline or in somatic cells. Xenopus laevis RAP55B has been shown to be part of translationally repressed mRNP complexes in early oocytes. Together these findings suggest that this protein family has evolved a common and fundamental role in the control of mRNA translation. Furthermore human RAP55A is an autoantigen detected in the serum of patients with primary biliary cirrhosis (PBC). The link between RAP55A, P-bodies and PBC remains to be elucidated.