A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing.

Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturation-inducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation.

Identification of embryonic RNA granules that act as sites of mRNA translation after changing their physical properties.

Fertilized eggs begin to translate mRNAs at appropriate times and placements to control development, but how the translation is regulated remains unclear. Here, we found that pou5f3 mRNA encoding a transcriptional factor essential for development formed granules in a dormant state in zebrafish oocytes. Although the number of pou5f3 granules remained constant, Pou5f3 protein accumulated after fertilization. Intriguingly, signals of newly synthesized peptides and a ribosomal protein became colocalized with pou5f3 granules after fertilization and, moreover, nascent Pou5f3 was shown to be synthesized in the granules. This functional change was accompanied by changes in the state and internal structure of granules. Dissolution of the granules reduced the rate of protein synthesis. Similarly, nanog and sox19b mRNAs in zebrafish and Pou5f1/Oct4 mRNA in mouse assembled into granules. Our results reveal that subcellular compartments, termed embryonic RNA granules, function as activation sites of translation after changing physical properties for directing vertebrate development.

Valosin-containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations.

Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.

Transgenic zebrafish reveals novel mechanisms of translational control of cyclin B1 mRNA in oocytes.

Temporal translation control of localized mRNA is crucial for regulating various cellular and developmental processes. However, little is known about the mechanisms of temporal translation control of localized mRNA due to the limitation in technology. cyclin B1 mRNA at the animal polar cytoplasm of immature zebrafish oocytes is translationally repressed, and its activation is temporally regulated during maturation. Mechanisms of cyclin B1 translation in oocytes were analyzed using transgenic zebrafish in which reporter mRNAs are produced from transgenes introduced into the genome through transcription in the nucleus followed by transport to the cytoplasm, as in endogenous mRNAs. Real-time imaging of the site and timing of translation showed that mRNAs containing the full-length cyclin B1 sequence precisely mimic the localization and translation of endogenous cyclin B1 mRNA. However, mRNAs containing cyclin B1 3' untranslated region but lacking open reading frame (ORF) underwent abnormal localization and precocious translational activation, indicating the significance of the ORF in translational control of cyclin B1 mRNA. Our genetic approach in combination with real-time imaging of the translation site and timing provides a novel insight into the mechanisms of temporal control of translation.

Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport.

Localization of RNAs to various subcellular destinations is a widely used mechanism that regulates a large proportion of transcripts in polarized cells. In many cases, such localized transcripts mediate spatial control of gene expression by being translationally silent while in transit and locally activated at their destination. Here, we investigate the translation of RNAs localized at dynamic cellular protrusions of human and mouse, migrating, mesenchymal cells. In contrast to the model described above, we find that protrusion-localized RNAs are not locally activated solely at protrusions, but can be translated with similar efficiency in both internal and peripheral locations. Interestingly, protrusion-localized RNAs are translated at extending protrusions, they become translationally silenced in retracting protrusions and this silencing is accompanied by coalescence of single RNAs into larger heterogeneous RNA clusters. This work describes a distinct mode of translational regulation of localized RNAs, which we propose is used to regulate protein activities during dynamic cellular responses.

Linking substrate and nucleus via actin cytoskeleton in pluripotency maintenance of mouse embryonic stem cells.

Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process.

FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination.

Cytoplasmic inclusions of the RNA-binding protein fused in sarcoma (FUS) represent one type of membraneless ribonucleoprotein compartment. Formation of FUS inclusions is promoted by amyotrophic lateral sclerosis (ALS)-linked mutations, but the cellular functions affected upon inclusion formation are poorly defined. In this study, we find that FUS inclusions lead to the mislocalization of specific RNAs from fibroblast cell protrusions and neuronal axons. This is mediated by recruitment of kinesin-1 mRNA and protein within FUS inclusions, leading to a loss of detyrosinated glutamate (Glu)-microtubules (MTs; Glu-MTs) and an inability to support the localization of RNAs at protrusions. Importantly, dissolution of FUS inclusions using engineered Hsp104 disaggregases, or overexpression of kinesin-1, reverses these effects. We further provide evidence that kinesin-1 affects MT detyrosination not through changes in MT stability, but rather through targeting the tubulin carboxypeptidase enzyme onto specific MTs. Interestingly, other pathological inclusions lead to similar outcomes, but through apparently distinct mechanisms. These results reveal a novel kinesin-dependent mechanism controlling the MT cytoskeleton and identify loss of Glu-MTs and RNA mislocalization as common outcomes of ALS pathogenic mutations.

Dysregulated axonal RNA translation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease that has been associated with a diverse array of genetic changes. Prominent among these are mutations in RNA-binding proteins (RBPs) or repeat expansions that give rise to toxic RNA species. RBPs are additionally central components of pathologic aggregates that constitute a disease hallmark, suggesting that dysregulation of RNA metabolism underlies disease progression. In the context of neuronal physiology, transport of RNAs and localized RNA translation in axons are fundamental to neuronal survival and function. Several lines of evidence suggest that axonal RNA translation is a central process perturbed by various pathogenic events associated with ALS. Dysregulated translation of specific RNA groups could underlie feedback effects that connect and reinforce disease manifestations. Among such candidates are RNAs encoding proteins involved in the regulation of microtubule dynamics. Further understanding of axonally dysregulated RNA targets and of the feedback mechanisms they induce could provide useful therapeutic insights. WIREs RNA 2016, 7:589-603. doi: 10.1002/wrna.1352 For further resources related to this article, please visit the WIREs website.

Cyclin B1 mRNA translation is temporally controlled through formation and disassembly of RNA granules.

Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation.

The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules.

RNA localization pathways direct numerous mRNAs to distinct subcellular regions and affect many physiological processes. In one such pathway the tumor-suppressor protein adenomatous polyposis coli (APC) targets RNAs to cell protrusions, forming APC-containing ribonucleoprotein complexes (APC-RNPs). Here, we show that APC-RNPs associate with the RNA-binding protein Fus/TLS (fused in sarcoma/translocated in liposarcoma). Fus is not required for APC-RNP localization but is required for efficient translation of associated transcripts. Labeling of newly synthesized proteins revealed that Fus promotes translation preferentially within protrusions. Mutations in Fus cause amyotrophic lateral sclerosis (ALS) and the mutant protein forms inclusions that appear to correspond to stress granules. We show that overexpression or mutation of Fus results in formation of granules, which preferentially recruit APC-RNPs. Remarkably, these granules are not translationally silent. Instead, APC-RNP transcripts are translated within cytoplasmic Fus granules. These results unexpectedly show that translation can occur within stress-like granules. Importantly, they identify a new local function for cytoplasmic Fus with implications for ALS pathology.