Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3′ UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1,473 in the 3′ UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.     

In cultured oligodendrocytes the A/B-type hnRNP CBF-A accompanies MBP mRNA bound to mRNA trafficking sequences

Heterogeneous ribonucleoproteins (hnRNPs) have key roles in RNA biogenesis, including pre-mRNP assembly, transport and cytoplasmic localization. Here we show by biochemical fractionation of nuclear extracts and protein-protein interaction assays that the A/B-type hnRNP CBF-A is in a multiprotein complex with hnRNP A2 and A3 and hnRNP U. Using RNA affinity chromatography and gel retardation assays, CBF-A was found to bind directly to RNA trafficking sequences in the 3'-UTR of the myelin basic protein (MBP) mRNA. In primary oligodendrocytes, astrocytes, neurons, and mouse forebrain sections, CBF-A revealed a characteristic granular cytoplasmic distribution. In mouse forebrain CBF-A-positive granules were preferentially found in regions with loosely bundled myelin fibers. In cultured oligodendrocytes, CBF-A was found to be specifically associated with endogenous MBP mRNA and CBF-A gene silencing resulted in the retention of MBP granules in the cell body. Finally, immunoelectron microscopy in differentiating oligodendrocytes showed that CBF-A is located in cytoplasmic granules that are often associated with the cytoskeleton. The results suggest that CBF-A is a novel transacting factor required for cytoplasmic mRNA transport and localization.     

Regulation of RNA granules by FMRP and implications for neurological diseases

RNA granule formation, which can be regulated by RNA‐binding proteins (RBPs) such as fragile X mental retardation protein (FMRP), acts as a mechanism to control both the repression and subcellular localization of translation. Dysregulated assembly of RNA granules has been implicated in multiple neurological disorders, such as amyotrophic lateral sclerosis. Thus, it is crucial to understand the cellular pathways impinging upon granule assembly or disassembly. The goal of this review is to summarize recent advances in our understanding of the role of the RBP, FMRP, in translational repression underlying RNA granule dynamics, mRNA transport and localized. We summarize the known mechanisms of translational regulation by FMRP, the role of FMRP in RNA transport granules, fragile X granules and stress granules. Focusing on the emerging link between FMRP and stress granules, we propose a model for how hyperassembly and hypoassembly of RNA granules may contribute to neurological diseases.     

Two ZBP1 KH domains facilitate beta-actin mRNA localization,granule formation,and cytoskeletal attachment

Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.     

Myosin-Va mediates RNA distribution in primary fibroblasts from multiple organs

Myosin-Va has been shown to have multiple functions in a variety of cell types, including a role in RNA transport in neurons. Using primary cultures of cells from organs of young dilute-lethal (Myo5a(d-l)/Myo5a(d-l)) null mutant mice and wild-type controls, we show that in some, but not all, tissues, RNA distribution is dramatically different in the homozygous null mutant cells. The dependence of RNA localization on myosin-Va correlates with the relative abundance of the brain-specific splicing pattern of the myosin-Va tail. We also show that myosin-Va is involved in RNA localization soon after synthesis, because the effects of its absence are diminished for RNAs that are more than 30 min old. Finally, we show that localization of beta-actin mRNA is significantly changed by the absence of myosin-Va. These results suggest that myosin-Va is involved in a transient transport or tethering function in the perinuclear region.     

Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.     

The targeting of Xcat2 mRNA to the germinal granules depends on a cis-acting germinal granule localization element within the 3'UTR

The germ cell lineage is specified by the germ plasm, which in Xenopus laevis contains putative determinants called germinal granules. The pathway through which these structures form and how their components are assembled remain unclear. Using a combination of electron microscopy and in situ hybridization with the germinal granule-associated Xcat2 mRNA we demonstrated that the granules were derived from a branching network of granulofibrillar material within the mitochondrial cloud. Targeting of Xcat2 mRNA to the germinal granules depended on a 164-nt 3'UTR germinal granule localization element (GGLE; nt 631-795) that was distinct from the previously defined mitochondrial cloud localization element (MCLE; nt 403-630; Y. Zhou and M. L. King, 1996, Development 122, 2947-2953). This demonstrated that the Xcat 3'UTR contains a compound localization element consisting of a general element (MCLE) targeting the RNA to the mitochondrial cloud and a second element (GGLE) responsible for targeting to the germinal granules within the cloud. The GGLE when fused to Xlsirt RNA was sufficient to target this nongranule mitochondrial cloud-associated RNA to the germinal granules. This is the first example of a localization element involved in targeting an mRNA to a specific subcellular target such as the germinal granules and suggests that cis-acting elements on RNAs play an important role in the assembly of germinal granules and, therefore, the establishment of the germ cell lineage.     

MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.     

A Comparative Quantitative Assessment of Axonal and Dendritic mRNA Transport in Maturing Hippocampal Neurons

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.     

Cytoplasmic organelles on the road to mRNA decay

Localization of both mRNAs and mRNA decay factors to internal membranes of eukaryotic cells provides a means of coordinately regulating mRNAs with common functions as well as coupling organelle function to mRNA turnover. The classic mechanism of mRNA localization to membranes is the signal sequence-dependent targeting of mRNAs encoding membrane and secreted proteins to the cytoplasmic surface of the endoplasmic reticulum. More recently, however, mRNAs encoding proteins with cytosolic or nuclear functions have been found associated with various organelles, in many cases through unknown mechanisms. Furthermore, there are several types of RNA granules, many of which are sites of mRNA degradation; these are frequently found associated with membrane-bound organelles such as endosomes and mitochondria. In this review we summarize recent findings that link organelle function and mRNA localization to mRNA decay. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

RNA Granule Protein 140 (RNG140), a Paralog of RNG105 Localized to Distinct RNA Granules in Neuronal Dendrites in the Adult Vertebrate Brain

RNA granules mediate the transport and local translation of their mRNA cargoes, which regulate cellular processes such as stress response and neuronal synaptic plasticity. RNA granules contain specific RNA-binding proteins, including RNA granule protein 105 (RNG105), which is likely to participate in the transport and translation of mRNAs. In the present report, an RNG105 paralog, RNG140 is described. A homolog of RNG105/RNG140 is found in insects, echinoderms, and urochordates, whereas vertebrates have both of the two genes. RNG140 and RNG105 are similar in that both bind to mRNAs and inhibit translation in vitro, induce the formation of RNA granules, are most highly expressed in the brain, and are localized to dendritic RNA granules, part of which are accumulated at postsynapses. However, they differ in several characteristics; RNG105 is highly expressed in embryonic brains, whereas RNG140 is highly expressed in adult brains. Furthermore, the granules where RNG105 or RNG140 is localized are distinct RNA granules in both cultured cells and neuronal dendrites. Thus, RNG140 is an RNA-binding protein that shows different expression and localization patterns from RNG105. Knockdown experiments in cultured neurons also are performed, which demonstrate that suppression of RNG140 or RNG105 reduces dendrite length and spine density. Knockdown effects of RNG140 were not rescued by RNG105, and vise versa, suggesting distinct roles of RNG105 and RNG140. These results suggest that RNG140 has roles in the maintenance of the dendritic structure in the adult vertebrate brain through localizing to a kind of RNA granules that are distinct from RNG105-containing granules.     

The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neurons

In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen2(62) isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen2(59) and Staufen2(52) in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen2(62), but not the Staufen2(59), isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen2(62)-containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.     

RNA localization in the cytoplasm

RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated. In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity. In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process.