scholarly journals Two- and three-dimensional tracking of MFA2 mRNA molecules in mating yeast

2020 ◽  
Author(s):  
Polina Geva ◽  
Konstantin Komoshvili ◽  
Stella Liberman-Aronov
Keyword(s):  
Molecular Motors ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Three Dimensional ◽  
Movement Behavior ◽  
Mrna Transport ◽  
Processing Bodies ◽  
Temporal Regulation ◽  
Spatio Temporal ◽  
Processing Body

AbstractIntracellular mRNA transport contributes to the spatio-temporal regulation of mRNA function and localized translation. In the budding yeast, Saccharomyces cerevisiae, asymmetric mRNA transport localizes ∼30 specific mRNAs including those encoding polarity and secretion factors, to the bud tip. The underlying process involves RNA binding proteins (RBPs), molecular motors, processing bodies (PBs), and the actin cytoskeleton. Recently, pheromone a-factor expression in mating yeast was discovered to depend upon proper localization of its mRNA, MFA2. MFA2 mRNAs in conjunction with PBs cluster at the shmoo tip to form “mating bodies”, from which a-factor is locally expressed. The mechanism ensuring the correct targeting of mRNA to the shmoo tip is poorly understood.Here we analyzed the kinetics and trajectories of MFA2 mRNA transport in living, alpha-factor treated yeast. Two-(2D) and three-dimensional (3D) analyses allowed us to reconstruct the granule tracks and estimate granule velocities. Tracking analysis of single MFA2 mRNA granules, labeled using a fluorescent aptamer system, demonstrated three types movement: vibrational, oscillatory and translocational. The mRNA granule transport was complex; a granule could change its movement behavior and composition during its journey to the shmoo. Processing body assembly and the actin-based motor, Myo4p, were involved in movement of MFA2 mRNA to the shmoo, but neither was required, indicating that multiple mechanisms for translocation were at play. Our visualization studies present a dynamic view of the localization mechanism in shmoo-bearing cells.

scholarly journals Two- and Three-Dimensional Tracking of MFA2 mRNA Molecules in Mating Yeast

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2151
Author(s):  
Polina Geva ◽  
Konstantin Komoshvili ◽  
Stella Liberman-Aronov
Keyword(s):  
Molecular Motors ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Three Dimensional ◽  
Movement Behavior ◽  
Mrna Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temporal Regulation ◽  
Spatio Temporal ◽  
Processing Body

Intracellular mRNA transport contributes to the spatio-temporal regulation of mRNA function and localized translation. In the budding yeast, Saccharomyces cerevisiae, asymmetric mRNA transport localizes ~30 specific mRNAs including those encoding polarity and secretion factors, to the bud tip. The underlying process involves RNA-binding proteins (RBPs), molecular motors, processing bodies (PBs), and the actin cytoskeleton. Recently, pheromone a-factor expression in mating yeast was discovered to depend on proper localization of its mRNA, MFA2 mRNAs in conjunction with PBs cluster at the shmoo tip to form “mating bodies”, from which a-factor is locally expressed. The mechanism ensuring the correct targeting of mRNA to the shmoo tip is poorly understood. Here we analyzed the kinetics and trajectories of MFA2 mRNA transport in living, alpha-factor treated yeast. Two- (2D) and three-dimensional (3D) analyses allowed us to reconstruct the granule tracks and estimate granule velocities. Tracking analysis of single MFA2 mRNA granules, labeled using a fluorescent aptamer system, demonstrated three types movement: vibrational, oscillatory and translocational. The mRNA granule transport was complex; a granule could change its movement behavior and composition during its journey to the shmoo. Processing body assembly and the actin-based motor, Myo4p, were involved in movement of MFA2 mRNA to the shmoo, but neither was required, indicating that multiple mechanisms for translocation were at play. Our visualization studies present a dynamic view of the localization mechanism in shmoo-bearing cells.

scholarly journals Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aidan M. Fenix ◽  
Yuichiro Miyaoka ◽  
Alessandro Bertero ◽  
Steven M. Blue ◽  
Matthew J. Spindler ◽  
...  
Keyword(s):  
Rna Binding ◽  
Acute Stress ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Circular Rna ◽  
Missense Mutations ◽  
Gain Of Function ◽  
Processing Bodies ◽  
Gene Splicing ◽  
Processing Body

AbstractMutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

scholarly journals Microtubule-Dependent mRNA Transport in Fungi

2010 ◽  
Vol 9 (7) ◽  
pp. 982-990 ◽  
Author(s):  
Kathi Zarnack ◽  
Michael Feldbrügge
Keyword(s):  
Molecular Motors ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Hyphal Growth ◽  
Subcellular Targeting ◽  
Mrna Transport ◽  
Long Distance ◽  
Content Type ◽  
Long Distance Transport

ABSTRACT The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen Ustilago maydis. Combining in vivo UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of U. maydis as a model system to uncover basic concepts of long-distance mRNA transport.

Roles for myosin Va in RNA transport and turnover

2012 ◽  
Vol 40 (6) ◽  
pp. 1416-1420 ◽  
Author(s):  
Mary W. McCaffrey ◽  
Andrew J. Lindsay
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Class V ◽  
Processing Bodies ◽  
Surface Receptors ◽  
P Bodies ◽  
Myosin Va ◽  
Myosin Vb ◽  
Actin Cables ◽  
The Brain

Mammals express three class V myosins. Myosin Va is widely expressed, but enriched in the brain, testes and melanocytes, myosin Vb is expressed ubiquitously, and myosin Vc is believed to be epithelium-specific. Myosin Va is the best characterized of the three and plays a key role in the transport of cargo to the plasma membrane. Its cargo includes cell-surface receptors, pigment and organelles such as the endoplasmic reticulum. It is also emerging that RNA and RNA-BPs (RNA-binding proteins) make up another class of myosin Va cargo. It has long been established that the yeast class V myosin, Myo4p, transports mRNAs along actin cables into the growing bud, and now several groups have reported a similar role for class V myosins in higher eukaryotes. Myosin Va has also been implicated in the assembly and maintenance of P-bodies (processing bodies), cytoplasmic foci that are involved in mRNA storage and degradation. The present review examines the evidence that myosin Va plays a role in the transport and turnover of mRNA.

scholarly journals ALY RNA-Binding Proteins Are Required for Nucleocytosolic mRNA Transport and Modulate Plant Growth and Development

2018 ◽  
Vol 177 (1) ◽  
pp. 226-240 ◽  
Author(s):  
Christina Pfaff ◽  
Hans F. Ehrnsberger ◽  
María Flores-Tornero ◽  
Brian B. Sørensen ◽  
Thomas Schubert ◽  
...  
Keyword(s):  
Plant Growth ◽  
Growth And Development ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Plant Growth And Development ◽  
Mrna Transport

scholarly journals First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene

2020 ◽  
Vol 21 (20) ◽  
pp. 7803
Author(s):  
Julie Miro ◽  
Anne-Laure Bougé ◽  
Eva Murauer ◽  
Emmanuelle Beyne ◽  
Dylan Da Cunha ◽  
...  
Keyword(s):  
Large Scale ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Dystrophin Gene ◽  
Rna Seq ◽  
Spatio Temporal ◽  
Dmd Gene ◽  
Shed Light

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

scholarly journals CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function

2016 ◽  
Vol 310 (1) ◽  
pp. C54-C65 ◽  
Author(s):  
Ting-Xi Yu ◽  
Bei-Lin Gu ◽  
Jun-Kai Yan ◽  
Jie Zhu ◽  
Wei-Hui Yan ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intestinal Barrier ◽  
Primary Component ◽  
Epithelial Barrier ◽  
Barrier Dysfunction ◽  
Processing Bodies ◽  
Epithelial Barrier Function ◽  
Epithelial Barrier Dysfunction ◽  
E Cadherin

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3′-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

scholarly journals Competitive binding of CUGBP1 and HuR to occludin mRNA controls its translation and modulates epithelial barrier function

2013 ◽  
Vol 24 (2) ◽  
pp. 85-99 ◽  
Author(s):  
Ting-Xi Yu ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
Lan Xiao ◽  
...  
Keyword(s):  
Barrier Function ◽  
Untranslated Region ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Monolayer ◽  
Intestinal Epithelial ◽  
Processing Bodies ◽  
Epithelial Barrier Function ◽  
P Bodies ◽  
The Stability

RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in epithelial tissues and modulate the stability and translation of target mRNAs. Here we present evidence that CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction (TJ) integrity in the intestinal epithelial cell monolayer. CUGBP1 and HuR competed for association with the same occludin 3′-untranslated region element and regulated occludin translation competitively and in opposite directions. CUGBP1 overexpression decreased HuR binding to occludin mRNA, repressed occludin translation, and compromised the TJ barrier function, whereas HuR overexpression inhibited CUGBP1 association with occludin mRNA and promoted occludin translation, thereby enhancing the barrier integrity. Repression of occludin translation by CUGBP1 was due to the colocalization of CUGBP1 and tagged occludin RNA in processing bodies (P-bodies), and this colocalization was prevented by HuR overexpression. These findings indicate that CUGBP1 represses occludin translation by increasing occludin mRNA recruitment to P-bodies, whereas HuR promotes occludin translation by blocking occludin mRNA translocation to P-bodies via the displacement of CUGBP1.

scholarly journals miR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies

2012 ◽  
Vol 23 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Yu-Hong Cui ◽  
Lan Xiao ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
Coding Region ◽  
Processing Bodies ◽  
Target Mrnas ◽  
Repressive Effect

microRNAs (miRNAs) and RNA-binding proteins (RBPs) jointly regulate gene expression at the posttranscriptional level and are involved in many aspects of cellular functions. The RBP CUG-binding protein 1 (CUGBP1) destabilizes and represses the translation of several target mRNAs, but the exact mechanism that regulates CUGBP1 abundance remains elusive. In this paper, we show that miR-503, computationally predicted to associate with three sites of the CUGBP1 mRNA, represses CUGBP1 expression. Overexpression of an miR-503 precursor (pre-miR-503) reduced the de novo synthesis of CUGBP1 protein, whereas inhibiting miR-503 by using an antisense RNA (antagomir) enhanced CUGBP1 biosynthesis and elevated its abundance; neither intervention changed total CUGBP1 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-503 through the CUGBP1 coding region sites than through the single CUGBP1 3′-untranslated region target site. CUGBP1 mRNA levels in processing bodies (P-bodies) increased in cells transfected with pre-miR-503, while silencing P-body resident proteins Ago2, RCK, or LSm4 decreased miR-503–mediated repression of CUGBP1 expression. Decreasing the levels of cellular polyamines reduced endogenous miR-503 levels and promoted CUGBP1 expression, an effect that was prevented by ectopic miR-503 overexpression. Repression of CUGBP1 by miR-503 in turn altered the expression of CUGBP1 target mRNAs and thus increased the sensitivity of intestinal epithelial cells to apoptosis. These findings identify miR-503 as both a novel regulator of CUGBP1 expression and a modulator of intestinal epithelial homoeostasis.

scholarly journals Single-molecule imaging of mRNA localization and regulation during the integrated stress response

2018 ◽  
Author(s):  
Johannes H. Wilbertz ◽  
Franka Voigt ◽  
Ivana Horvathova ◽  
Gregory Roth ◽  
Yinxiu Zhan ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Molecule ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Localization ◽  
Mrna Degradation ◽  
Integrated Stress Response ◽  
Cellular Space ◽  
Processing Bodies ◽  
Functional Consequence

AbstractBiological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules(SGs) and processing bodies(PBs), which are distinct biomolecular condensates, and its effect on translation and mRNA degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics and that specific RNA binding proteins can anchor mRNAs within these compartments. During recovery from stress, mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts.

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