RNA Binding Protein TAF15 Suppresses Toxicity in a Yeast Model of FUS Proteinopathy

Abstract Mutations in Fused in Sarcoma (FUS), an RNA binding protein that functions in multiple steps in gene expression regulation and RNA processing, are known to cause familial amyotrophic lateral sclerosis (ALS). Since this discovery, mutations in several other RNA binding proteins (RBPs) have also been linked to ALS. Some of these ALS-associated RBPs have been shown to colocalize with ribonucleoprotein (RNP) granules such as stress granules and processing bodies (p-bodies). Characterization of ALS-associated proteins, their mis-localization, aggregation and toxicity in cellular and animal models have provided critical insights in disease. More and more evidence has emerged supporting a hypothesis that impaired clearance, inappropriate assembly, and dysregulation of RNP granules play a role in ALS. Through genome-scale overexpression screening of a yeast model of FUS toxicity, we found that TAF15, a human RBP with a similar protein domain structure and belonging to the same FET protein family as FUS, suppresses FUS toxicity. The suppressor effect of TAF15 is specific to FUS and not found in other yeast models of neurodegenerative disease-associated proteins. We showed that the RNA recognition motif (RRM) of TAF15 is required for its rescue of FUS toxicity. Furthermore, FUS and TAF15 physically interact, and the C-terminus of TAF15 is required for both the physical protein-protein interaction and its protection against FUS toxicity. Finally, while FUS induces and colocalizes with both stress granules and p-bodies, TAF15 only induces and colocalizes with p-bodies. Importantly, co-expression of FUS and TAF15 induces more p-bodies than individually expressing each gene alone, and FUS toxicity is exacerbated in yeast that is deficient in p-body formation. Overall, our findings suggest a role of p-body formation in the suppression of FUS toxicity by TAF15.

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The Putative RNA-Binding Protein Nrp1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered Into Heat-Induced Protein Aggregates in Fission Yeast

10.20944/preprints202103.0452.v1 ◽

2021 ◽

Author(s):

Masashi Yukawa ◽

Mitsuki Ohishi ◽

Yusuke Yamada ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Protein Aggregates ◽

Associated Proteins ◽

Spindle Microtubules ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here we show that deletion of the nrp1 gene, which encodes a putative RNA-binding protein with unknown function, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the nrp1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Nrp1 are essential for its cytoplasmic localization and function. We have also found that a portion of Nrp1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Nrp1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Quarterly Reviews of Biophysics ◽

10.1017/s0033583515000207 ◽

2015 ◽

Vol 49 ◽

Cited By ~ 21

Author(s):

Marianna Teplova ◽

Thalia A. Farazi ◽

Thomas Tuschl ◽

Dinshaw J. Patel

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Hek293 Cells ◽

Structural Basis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Muscle Plasticity ◽

Rrm Domain

AbstractRNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

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Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3494 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3494-3504 ◽

Cited By ~ 238

Author(s):

T D Levine ◽

F Gao ◽

P H King ◽

L G Andrews ◽

J D Keene

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Selection Procedure ◽

Vital Role ◽

Untranslated Regions ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Recognition Motifs

We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

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Poly(A) binding protein is required for mRNP remodeling to form P-bodies in mammalian cells

10.1101/2021.02.07.430159 ◽

2021 ◽

Author(s):

Jingwei Xie ◽

Yu Chen ◽

Xiaoyu Wei ◽

Guennadi Kozlov

Keyword(s):

Mammalian Cells ◽

Binding Protein ◽

Stress Granules ◽

Body Formation ◽

P Bodies ◽

Rna Granules ◽

Cellular Processes ◽

Early Stages ◽

Summary Statement

AbstractCompartmentalization of mRNA through formation of RNA granules is involved in many cellular processes, yet it is not well understood. mRNP complexes undergo dramatic changes in protein compositions, reflected by markers of P-bodies and stress granules. Here, we show that PABPC1, albeit absent in P-bodies, plays important role in P-body formation. Depletion of PABPC1 decreases P-body population in unstressed cells. Upon stress in PABPC1 depleted cells, individual P-bodies fail to form and instead P-body proteins assemble on PABPC1-containing stress granules. We hypothesize that mRNP recruit proteins via PABPC1 to assemble P-bodies, before PABPC1 is displaced from mRNP. Further, we demonstrate that GW182 can mediate P-body assembly. These findings help us understand the early stages of mRNP remodeling and P-body formation.Summary statementA novel role of poly(A) binding protein is reported in P-body formation

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KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182

Nucleic Acids Research ◽

10.1093/nar/gkz683 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9368-9385 ◽

Cited By ~ 5

Author(s):

Nishi R Sharma ◽

Vladimir Majerciak ◽

Michael J Kruhlak ◽

Lulu Yu ◽

Jeong Gu Kang ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Time Lapse ◽

Processing Bodies ◽

P Bodies ◽

Rna Granules ◽

Inhibitory Function ◽

Invasion Mechanisms ◽

Initial Stage

Abstract Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

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LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00332-07 ◽

2007 ◽

Vol 27 (18) ◽

pp. 6469-6483 ◽

Cited By ~ 172

Author(s):

John L. Goodier ◽

Lili Zhang ◽

Melissa R. Vetter ◽

Haig H. Kazazian

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Protein A ◽

Rna Binding Protein ◽

Stress Granules ◽

Plasminogen Activator Inhibitor ◽

Cellular Damage ◽

Activator Inhibitor

ABSTRACT LINE-1 retrotransposons constitute one-fifth of human DNA and have helped shape our genome. A full-length L1 encodes a 40-kDa RNA-binding protein (ORF1p) and a 150-kDa protein (ORF2p) with endonuclease and reverse transcriptase activities. ORF1p is distinctive in forming large cytoplasmic foci, which we identified as cytoplasmic stress granules. A phylogenetically conserved central region of the protein is critical for wild-type localization and retrotransposition. Yeast two-hybrid screens revealed several RNA-binding proteins that coimmunoprecipitate with ORF1p and colocalize with ORF1p in foci. Two of these proteins, YB-1 and hnRNPA1, were previously reported in stress granules. We identified additional proteins associated with stress granules, including DNA-binding protein A, 9G8, and plasminogen activator inhibitor RNA-binding protein 1 (PAI-RBP1). PAI-RBP1 is a homolog of VIG, a part of the Drosophila melanogaster RNA-induced silencing complex (RISC). Other RISC components, including Ago2 and FMRP, also colocalize with PAI-RBP1 and ORF1p. We suggest that targeting ORF1p, and possibly the L1 RNP, to stress granules is a mechanism for controlling retrotransposition and its associated genetic and cellular damage.

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Glycine-Rich RNA-Binding Protein 7 interacts with and potentiates effector-induced immunity by Gpa2 and Rx1 based on an intact RNA Recognition Motif

10.1101/2021.05.18.444452 ◽

2021 ◽

Author(s):

Octavina CA Sukarta ◽

Qi Zheng ◽

Erik J Slootweg ◽

Mark Mekken ◽

Melanie Mendel ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Potato Virus X ◽

Globodera Pallida ◽

Transcriptional Level ◽

Rna Recognition Motif ◽

Rna Recognition ◽

In Planta ◽

Recognition Motif

The activity of intracellular plant Nucleotide-Binding Leucine-Rich Repeat (NB-LRR) immune receptors is fine-tuned by interactions between the receptors and their partners. Identifying NB-LRR interacting proteins is, therefore, crucial to advance our understanding of how these receptors function. A Co-Immunoprecipitation/Mass-Spectrometry screening was performed in Nicotiana benthamiana to identify host proteins associated with the Gpa2 CC-NB-LRR, which confers resistance against the potato cyst nematode Globodera pallida. A combination of biochemical, cellular, and functional assays was used to assess the role of a candidate interactor in defence. A N. benthamiana homolog of the Glycine-Rich RNA-Binding Protein 7 (NbGRP7) protein was prioritized as a novel Gpa2-interacting protein for further investigations. NbGRP7 also associates in planta with the homologous Rx1 receptor, which confers immunity to Potato Virus X. We show that NbGRP7 positively regulates extreme resistance by Rx1 and cell death by Gpa2. Mutating the NbGRP7 RNA recognition motif compromises its role in Rx1-mediated defence. Strikingly, ectopic NbGRP7 expression impacts the steady-state levels of Rx1, which relies on an intact RNA recognition motif. Combined, our findings illustrate that NbGRP7 is a novel pro-immune component in effector-triggered immunity by regulating Gpa2/Rx1 functioning at a post-transcriptional level.

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Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3494-3504.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3494-3504

Author(s):

T D Levine ◽

F Gao ◽

P H King ◽

L G Andrews ◽

J D Keene

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Selection Procedure ◽

Vital Role ◽

Untranslated Regions ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Recognition Motifs

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RNA-recognition motif in Matrin-3 mediates neurodegeneration through interaction with hnRNPM

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01021-5 ◽

2020 ◽

Vol 8 (1) ◽

Cited By ~ 2

Author(s):

Nandini Ramesh ◽

Sukhleen Kour ◽

Eric N. Anderson ◽

Dhivyaa Rajasundaram ◽

Udai Bhan Pandey

Keyword(s):

Motor Neurons ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Dependent Manner ◽

Recognition Motif ◽

Matrin 3

Abstract Background Amyotrophic lateral sclerosis (ALS) is an adult-onset, fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons. While pathogenic mutations in the DNA/RNA-binding protein Matrin-3 (MATR3) are linked to ALS and distal myopathy, the molecular mechanisms underlying MATR3-mediated neuromuscular degeneration remain unclear. Methods We generated Drosophila lines with transgenic insertion of human MATR3 wildtype, disease-associated variants F115C and S85C, and deletion variants in functional domains, ΔRRM1, ΔRRM2, ΔZNF1 and ΔZNF2. We utilized genetic, behavioral and biochemical tools for comprehensive characterization of our models in vivo and in vitro. Additionally, we employed in silico approaches to find transcriptomic targets of MATR3 and hnRNPM from publicly available eCLIP datasets. Results We found that targeted expression of MATR3 in Drosophila muscles or motor neurons shorten lifespan and produces progressive motor defects, muscle degeneration and atrophy. Strikingly, deletion of its RNA-recognition motif (RRM2) mitigates MATR3 toxicity. We identified rump, the Drosophila homolog of human RNA-binding protein hnRNPM, as a modifier of mutant MATR3 toxicity in vivo. Interestingly, hnRNPM physically and functionally interacts with MATR3 in an RNA-dependent manner in mammalian cells. Furthermore, common RNA targets of MATR3 and hnRNPM converge in biological processes important for neuronal health and survival. Conclusions We propose a model of MATR3-mediated neuromuscular degeneration governed by its RNA-binding domains and modulated by interaction with splicing factor hnRNPM.

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

The Journal of Cell Biology ◽

10.1083/jcb.201306058 ◽

2013 ◽

Vol 203 (5) ◽

pp. 737-746 ◽

Cited By ~ 102

Author(s):

Kyota Yasuda ◽

Huaye Zhang ◽

David Loiselle ◽

Timothy Haystead ◽

Ian G. Macara ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Localization ◽

Polyposis Coli ◽

Fused In Sarcoma ◽

Ribonucleoprotein Complexes ◽

Physiological Processes ◽

Rnp Granules ◽

Lateral Sclerosis

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.

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