Cis-acting determinants of asymmetric, cytoplasmic RNA transport

Asymmetric subcellular distribution of RNA is used by many organisms to establish cell polarity, differences in cell fate, or to sequester protein activity. Accurate localization of RNA requires specific sequence and/or structural elements in the localized RNA, as well as proteins that recognize these elements and link the RNA to the appropriate molecular motors. Recent advances in biochemistry, molecular biology, and cell imaging have enabled the identification of many RNA localization elements, or "zipcodes," from a variety of systems. This review focuses on the mechanisms by which various zipcodes direct RNA transport and on the known sequence/structural requirements for their recognition by transport complexes. Computational and experimental methods for predicting and identifying zipcodes are also discussed.     

A Xenopus protein related to hnRNP I has a role in cytoplasmic RNA localization.

Cytoplasmic localization of mRNA molecules is a powerful mechanism for generating cell polarity. In vertebrates, one paradigm is localization of Vg1 RNA within the Xenopus oocyte, a process directed by recognition of a localization element within the Vg1 3' UTR. We show that specific base changes within the localization element abolish both localization in vivo and binding in vitro by a single protein, VgRBP60. VgRBP60 is homologous to a human hnRNP protein, hnRNP I, and combined immunolocalization and in situ hybridization demonstrate striking colocalization of hnRNP I and Vg1 RNA within the vegetal cytoplasm of the Xenopus oocyte. These results implicate a novel role in cytoplasmic RNA transport for this family of nuclear RNA-binding proteins.     

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.     

Low-molecular-weight RNA of eukaryotes: biogenesis, subcellular localization, functions

The experimental data on the organization and expression of the low-molecular-weight RNA genes on the subcellular localization, metabolism and evolution of these RNA are summarized. The processes in which they are involved are discussed. The biological role of the low-molecular-weight RNA is paid a special attention. A hypothesis concerning the involvement of these molecules in the mechanism of active transport of proteins from the cytoplasm into the nucleus of the cell is put forward.     

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the ?1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.     

Cell polarity and actin mRNA localization

In many species, intracellular mRNA localization is linked to cell polarity. In many cases however, mRNAs become localized as a result of a pre-existing cell-polarity, and they do not modify it. Remarkably, in the case beta actin mRNA in vertebrate, it has been shown that the transport and localization of this RNA is required for the establishment and maintenance of cell polarity. This occurs in fibroblasts, but, very interestingly, in immature neurons as well. This review will describe the functions and mechanisms of actin mRNA localization.     

In vivo visualization of RNA in plants cells using the λN₂₂ system and a GATEWAY-compatible vector series for candidate RNAs

The past decade has seen a tremendous increase in RNA research, which has demonstrated that RNAs are involved in many more processes than were previously thought. The dynamics of RNA synthesis towards their regulated activity requires the interplay of RNAs with numerous RNA binding proteins (RBPs). The localization of RNA, a mechanism for controlling translation in a spatial and temporal fashion, requires processing and assembly of RNA into transport granules in the nucleus, transport towards cytoplasmic destinations and regulation of its activity. Compared with animal model systems little is known about RNA dynamics and motility in plants. Commonly used methods to study RNA transport and localization are time‐consuming, and require expensive equipment and a high level of experimental skill. Here, we introduce the λN₂₂ RNA stem‐loop binding system for the in vivo visualization of RNA in plant cells. The λN₂₂ system consists of two components: the λN₂₂ RNA binding peptide and the corresponding box‐B stem loops. We generated fusions of λN₂₂ to different fluorophores and a GATEWAY vector series for the simple fusion of any target RNA 5′ or 3′ to box‐B stem loops. We show that the λN₂₂ system can be used to detect RNAs in transient expression assays, and that it offers advantages compared with the previously described MS2 system. Furthermore, the λN₂₂ system can be used in combination with the MS2 system to visualize different RNAs simultaneously in the same cell. The toolbox of vectors generated for both systems is easy to use and promises significant progress in our understanding of RNA transport and localization in plant cells.     

Polar transport in the Drosophila oocyte requires Dynein and Kinesin I cooperation

BACKGROUND: The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS: Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION: The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.     

Axes formation and RNA localization

Axes formation in flies and frogs largely depends on RNA localization pathways functioning in the oocytes. It is thought that motors moving along the cytoskeleton enable the selective transport of RNAs to different destinations during oocyte development. Many of the steps in RNA localization are conserved, despite the existence of a variety of mechanisms, including the formation of nuclear ribonucleoprotein complexes, and active transport along microtubules.     

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.     

Ribonucleoprotein remodeling during RNA localization

Cytoplasmic RNA localization is a means to create polarity by restricting protein expression to a discrete subcellular location. RNA localization is a multistep process that begins with the recognition of cis-acting sequences within the RNA by specific trans-factors, and RNAs are localized in ribonucleoprotein (RNP) complexes that contain both the RNA and numerous protein components. Components of the localization machinery transport the RNP complex, usually in a translationally repressed state, to a distinct subcellular region, resulting in spatially restricted gene expression. Recent efforts to identify both the cis- and trans-factors required for RNA localization have elucidated RNA-protein interactions that are remodeled during localization.     

In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.     

Nuclear RNP complex assembly initiates cytoplasmic RNA localization

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA-protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.