Microtubule-Dependent mRNA Transport in Fungi

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.

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RNA Motifs and Modification Involve in RNA Long-Distance Transport in Plants

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.651278 ◽

2021 ◽

Vol 9 ◽

Author(s):

Tao Wang ◽

Xiaojun Li ◽

Xiaojing Zhang ◽

Qing Wang ◽

Wenqian Liu ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Future Research ◽

Rna Transport ◽

Long Distance ◽

Rna Motifs ◽

Related Sequence ◽

Long Distance Transport ◽

Nucleotide Mutation ◽

Distance Transport

A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling.

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A Posttranscriptional Mechanism That Controls Ptbp1 Abundance in the Xenopus Epidermis

Molecular and Cellular Biology ◽

10.1128/mcb.01040-14 ◽

2014 ◽

Vol 35 (4) ◽

pp. 758-768 ◽

Cited By ~ 4

Author(s):

Agnès Méreau ◽

Vincent Anquetil ◽

Hubert Lerivray ◽

Justine Viet ◽

Claire Schirmer ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Fine Tuning ◽

Splice Isoforms ◽

Content Type ◽

Splicing Pattern ◽

Exon 11 ◽

Splicing Patterns ◽

Different Tissues

The output of alternative splicing depends on the cooperative or antagonistic activities of several RNA-binding proteins (RBPs), like Ptbp1 and Esrp1 inXenopus. Fine-tuning of the RBP abundance is therefore of prime importance to achieve tissue- or cell-specific splicing patterns. Here, we addressed the mechanisms leading to the high expression of theptbp1gene, which encodes Ptbp1, inXenopusepidermis. Two splice isoforms ofptbp1mRNA differ by the presence of an alternative exon 11, and only the isoform including exon 11 can be translated to a full-length protein.In vivominigene assays revealed that the nonproductive isoform was predominantly produced. Knockdown experiments demonstrated that Esrp1, which is specific to the epidermis, strongly stimulated the expression ofptbp1by favoring the productive isoform. Consequently, knocking downesrp1phenocopiedptbp1inactivation. Conversely, Ptbp1 repressed the expression of its own gene by favoring the nonproductive isoform. Hence, a complex posttranscriptional mechanism controls Ptbp1 abundance inXenopusepidermis: skipping of exon 11 is the default splicing pattern, but Esrp1 stimulatesptbp1expression by favoring the inclusion of exon 11 up to a level that is limited by Ptbp1 itself. These results decipher a posttranscriptional mechanism that achieves various abundances of the ubiquitous RBP Ptbp1 in different tissues.

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Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

Eukaryotic Cell ◽

10.1128/ec.00018-14 ◽

2014 ◽

Vol 13 (5) ◽

pp. 664-674 ◽

Cited By ~ 19

Author(s):

Bhaskar Anand Jha ◽

Abeer Fadda ◽

Clementine Merce ◽

Elisha Mugo ◽

Dorothea Droll ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Open Reading Frames ◽

Mrna Levels ◽

Coding Region ◽

Content Type ◽

Puf Proteins ◽

Reading Frames

ABSTRACT Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

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

Cells ◽

10.3390/cells9102151 ◽

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.

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Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Journal of Bacteriology ◽

10.1128/jb.00343-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2458-2469 ◽

Cited By ~ 14

Author(s):

Kayley H. Schulmeyer ◽

Manisha R. Diaz ◽

Thomas B. Bair ◽

Wes Sanders ◽

Cindy J. Gode ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Posttranscriptional Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Consensus Sequence ◽

Stem Loop ◽

Content Type

ABSTRACTCsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level.Pseudomonas aeruginosahas a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed withtssA1, anin vivoregulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent.IMPORTANCEThe CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis.P. aeruginosahas two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution toP. aeruginosaphysiology and virulence.

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

The Journal of Cell Biology ◽

10.1083/jcb.201302095 ◽

2013 ◽

Vol 203 (6) ◽

pp. 971-984 ◽

Cited By ~ 19

Author(s):

Roland G. Heym ◽

Dennis Zimmermann ◽

Franziska T. Edelmann ◽

Lars Israel ◽

Zeynep Ökten ◽

...

Keyword(s):

Nuclear Export ◽

Messenger Rna ◽

Rna Binding ◽

Long Distance ◽

Long Distance Transport ◽

In Vitro Reconstitution ◽

Localization Elements ◽

Transport Complex

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.

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

10.1101/2020.07.02.185355 ◽

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.

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The multi PAM2 protein Upa2 functions as novel core component of endosomal mRNA transport

10.1101/455378 ◽

2018 ◽

Cited By ~ 1

Author(s):

Silke Jankowski ◽

Thomas Pohlmann ◽

Sebastian Baumann ◽

Kira Müntjes ◽

Senthil Kumar Devan ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Hyphal Growth ◽

Dependent Manner ◽

Mrna Transport ◽

Core Component ◽

Ribonucleoprotein Complexes ◽

Core Components ◽

Almost All ◽

Terminal Effector

AbstractmRNA transport determines spatiotemporal protein expression. Transport units are higher-order ribonucleoprotein complexes containing cargo mRNAs, RNA-binding proteins and accessory proteins. Endosomal mRNA transport in fungal hyphae belongs to the best-studied translocation mechanisms. Although several factors are known, additional core components are missing. Here, we describe the 232 kDa protein Upa2 containing multiple PAM2 motifs (poly[A]-binding protein [Pab1] associated motif 2) as a novel core component. Loss of Upa2 disturbs transport of cargo mRNAs and associated Pab1. Upa2 is present on almost all transport endosomes in an mRNA dependent-manner. Surprisingly, all four PAM2 motifs are dispensable for function during unipolar hyphal growth. Instead, Upa2 harbours a novel N-terminal effector domain as important functional determinant as well as a C-terminal GWW motif for specific endosomal localisation. In essence, Upa2 meets all the criteria of a novel core component of endosomal mRNA transport and appears to carry out crucial scaffolding functions.

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Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

Methods and Protocols ◽

10.3390/mps4010022 ◽

2021 ◽

Vol 4 (1) ◽

pp. 22

Author(s):

Mrinmoyee Majumder ◽

Viswanathan Palanisamy

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Patterns ◽

Control Of Gene Expression ◽

Regulatory Pathways ◽

Advantages And Disadvantages ◽

Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

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RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

Non-Coding RNA ◽

10.3390/ncrna7010011 ◽

2021 ◽

Vol 7 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

André P. Gerber

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Cell Protein ◽

Transcriptional Regulatory Networks ◽

Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

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