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

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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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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Microtubule-Dependent mRNA Transport in Fungi

Eukaryotic Cell ◽

10.1128/ec.00030-10 ◽

2010 ◽

Vol 9 (7) ◽

pp. 982-990 ◽

Cited By ~ 25

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.

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PUB1: a major yeast poly(A)+ RNA-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6114-6123.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6114-6123

Author(s):

M J Matunis ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Polymerase Ii ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Uv Light ◽

Binding Protein ◽

Gene Replacement ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Domains ◽

Development And Differentiation

The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins.

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

PLANT PHYSIOLOGY ◽

10.1104/pp.18.00173 ◽

2018 ◽

Vol 177 (1) ◽

pp. 226-240 ◽

Cited By ~ 13

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

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

International Journal of Molecular Sciences ◽

10.3390/ijms21207803 ◽

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.

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miRbiom: Machine-learning on Bayesian causal nets of RBP-miRNA interactions successfully predicts miRNA profiles

PLoS ONE ◽

10.1371/journal.pone.0258550 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258550

Author(s):

Upendra Kumar Pradhan ◽

Nitesh Kumar Sharma ◽

Prakash Kumar ◽

Ashwani Kumar ◽

Sagar Gupta ◽

...

Keyword(s):

Machine Learning ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mature Mirnas ◽

Learning System ◽

Mirna Biogenesis ◽

Rna Seq ◽

Human Mirnas ◽

Research Areas ◽

Spatio Temporal

Formation of mature miRNAs and their expression is a highly controlled process. It is very much dependent upon the post-transcriptional regulatory events. Recent findings suggest that several RNA binding proteins beyond Drosha/Dicer are involved in the processing of miRNAs. Deciphering of conditional networks for these RBP-miRNA interactions may help to reason the spatio-temporal nature of miRNAs which can also be used to predict miRNA profiles. In this direction, >25TB of data from different platforms were studied (CLIP-seq/RNA-seq/miRNA-seq) to develop Bayesian causal networks capable of reasoning miRNA biogenesis. The networks ably explained the miRNA formation when tested across a large number of conditions and experimentally validated data. The networks were modeled into an XGBoost machine learning system where expression information of the network components was found capable to quantitatively explain the miRNAs formation levels and their profiles. The models were developed for 1,204 human miRNAs whose accurate expression level could be detected directly from the RNA-seq data alone without any need of doing separate miRNA profiling experiments like miRNA-seq or arrays. A first of its kind, miRbiom performed consistently well with high average accuracy (91%) when tested across a large number of experimentally established data from several conditions. It has been implemented as an interactive open access web-server where besides finding the profiles of miRNAs, their downstream functional analysis can also be done. miRbiom will help to get an accurate prediction of human miRNAs profiles in the absence of profiling experiments and will be an asset for regulatory research areas. The study also shows the importance of having RBP interaction information in better understanding the miRNAs and their functional projectiles where it also lays the foundation of such studies and software in future.

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Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e13-01-0038 ◽

2013 ◽

Vol 24 (19) ◽

pp. 3069-3084 ◽

Cited By ~ 48

Author(s):

Judith Kraut-Cohen ◽

Evgenia Afanasieva ◽

Liora Haim-Vilmovsky ◽

Boris Slobodin ◽

Ido Yosef ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Single Molecule ◽

Translational Control ◽

Rna Binding ◽

Protein Translocation ◽

Rna Binding Proteins ◽

Signal Recognition Particle ◽

Dependent Manner ◽

Yeast Saccharomyces Cerevisiae ◽

Mrna Targeting

mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation- and signal recognition particle (SRP)–independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA–ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation- and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).

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A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8399 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8399-8407 ◽

Cited By ~ 109

Author(s):

J Flach ◽

M Bossie ◽

J Vogel ◽

A Corbett ◽

T Jinks ◽

...

Keyword(s):

Nuclear Protein ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Binding Protein ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Motifs ◽

C Terminus ◽

N Terminus ◽

Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

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Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei

Open Biology ◽

10.1098/rsob.160159 ◽

2017 ◽

Vol 7 (4) ◽

pp. 160159 ◽

Cited By ~ 4

Author(s):

Luis Miguel De Pablos ◽

Steve Kelly ◽

Janaina de Freitas Nascimento ◽

Jack Sunter ◽

Mark Carrington

Keyword(s):

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ectopic Expression ◽

Developmentally Regulated ◽

Mrna Metabolism ◽

Ribonucleoprotein Complexes ◽

Processing Body ◽

External Signals

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

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