scholarly journals The C-terminal RRM/ACT domain is crucial for fine-tuning the activation of ‘long’ RelA-SpoT Homolog enzymes by ribosomal complexes

2019 ◽  
Author(s):  
Hiraku Takada ◽  
Mohammad Roghanian ◽  
Victoriia Murina ◽  
Ievgen Dzhygyr ◽  
Rikinori Murayama ◽  
...  
Keyword(s):  
Stringent Response ◽  
Hydrolytic Activity ◽  
Chorismate Mutase ◽  
Fine Tuning ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Rrm Domain ◽  
Act Domain ◽  
Synthesis And Degradation ◽  
Ribosomal Complex

AbstractThe (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain ‘short’ and multi-domain ‘long’ RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA) / RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e. capable of both degrading and synthesising the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterised Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex – the ultimate inducer of (p)ppGpp production by RelA and Rel – and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.

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

2015 ◽  
Vol 49 ◽  
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.

scholarly journals The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans

2004 ◽  
Vol 167 (4) ◽  
pp. 639-647 ◽  
Author(s):  
Akwasi Anyanful ◽  
Kanako Ono ◽  
Robert C. Johnsen ◽  
Hinh Ly ◽  
Victor Jensen ◽  
...  
Keyword(s):  
Rna Binding ◽  
Functional Redundancy ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Speckled Pattern ◽  
Tissue Specific ◽  
C Elegans ◽  
Rrm Domain ◽  
New Family ◽  
Specific Alternative

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3′-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.

scholarly journals Structural basis for RNA recognition by a type II poly(A)-binding protein

2008 ◽  
Vol 105 (40) ◽  
pp. 15317-15322 ◽  
Author(s):  
Jikui Song ◽  
Jered V. McGivern ◽  
Karl W. Nichols ◽  
John L. Markley ◽  
Michael D. Sheets
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Recognition Site ◽  
Structural Basis ◽  
Rna Recognition Motif ◽  
Functional Domain ◽  
Rna Recognition ◽  
Type Ii ◽  
Rrm Domain ◽  
Polyproline Motif

We identified a functional domain (XlePABP2-TRP) of Xenopus laevis embryonic type II poly(A)-binding protein (XlePABP2). The NMR structure of XlePABP2-TRP revealed that the protein is a homodimer formed by the antiparallel association of β-strands from the single RNA recognition motif (RRM) domain of each subunit. In each subunit of the homodimer, the canonical RNA recognition site is occluded by a polyproline motif. Upon poly(A) binding, XlePABP2-TRP undergoes a dimer-monomer transition that removes the polyproline motif from the RNA recognition site and allows it to be replaced by the adenosine nucleotides of poly(A). Our results provide high-resolution structural information concerning type II PABPs and an example of a single RRM domain protein that transitions from a homodimer to a monomer upon RNA binding. These findings advance our understanding of RRM domain regulation, poly(A) recognition, and are relevant to understanding how type II PABPs function in mRNA processing and human disease.

A conserved RNA recognition motif (RRM) domain of Brassica napus FCA improves cotton fiber quality and yield by regulating cell size

2011 ◽  
Vol 30 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Fan Sun ◽  
Chuanliang Liu ◽  
Chaojun Zhang ◽  
Weiwei Qi ◽  
Xueyan Zhang ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Cell Size ◽  
Cotton Fiber ◽  
Fiber Quality ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Rrm Domain ◽  
Cotton Fiber Quality

Overexpression of the rFCA RNA Recognition Motif Affects Morphologies Modifications in Rice (Oryza sativa L.)

2007 ◽  
Vol 27 (4-5) ◽  
pp. 225-234 ◽  
Author(s):  
Fang Hong ◽  
Kotb Attia ◽  
Chun Wei ◽  
Kegui Li ◽  
Guangming He ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Oryza Sativa L ◽  
Constitutive Expression ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Interaction Domain ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Transgenic Lines ◽  
Recognition Motifs

RNA recognition motifs as important regulators of gene expression are highly conserved in animals and plants. The FCA floral promotion gene in Arabidopsis encodes a protein, containing two RNA recognition motifs (RRM) and a WW protein interaction domain. Here we isolated FCA cDNA from rice. FCA in rice (rFCA) was homologous to FCA-gamma of Arabidopsis and contained conserved domains. To investigate the function of RRM domain, fragment RRM1 and RRM2 of rFCA were introduced into rice subspecies Oryza sativa L. subsp. Indica var. 9311 and another rice subspecies Oryza sativa L. subsp. Japonica var. zhonghua11 transformation. Two transgenic lines exhibited similar phenotypes, flowering time delay, seed size and cell volume of transgenic plants was increased. These results showed that constitutive expression of RRMs could regulate cellular size. The patterns of overexpression of two RRM domains and their similar morphologies indicate they may play a same role.

scholarly journals The ACT domain in chloroplast precursor–phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity

2019 ◽  
Vol 294 (46) ◽  
pp. 17278-17288 ◽  
Author(s):  
Ahmed Eisa ◽  
Bettina Bölter ◽  
Serena Schwenkert
Keyword(s):  
Chorismate Mutase ◽  
Fine Tuning ◽  
Size Exclusion ◽  
Structural Motif ◽  
Aspartate Kinase ◽  
In Planta ◽  
Prephenate Dehydrogenase ◽  
Chloroplast Differentiation ◽  
Act Domain

Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo. Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.

Solution Structure of the Second RNA Recognition Motif (RRM) Domain of Murine T Cell Intracellular Antigen-1 (TIA-1) and Its RNA Recognition Mode†‡

Biochemistry ◽  
2008 ◽  
Vol 47 (24) ◽  
pp. 6437-6450 ◽  
Author(s):  
Kanako Kuwasako ◽  
Mari Takahashi ◽  
Naoya Tochio ◽  
Chikage Abe ◽  
Kengo Tsuda ◽  
...  
Keyword(s):  
T Cell ◽  
Solution Structure ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Rrm Domain ◽  
Intracellular Antigen

scholarly journals RRM domain of ALS/FTD-causing FUS interacts with membrane: an anchor of membraneless organelles to membranes?

2017 ◽  
Author(s):  
Yimei Lu ◽  
Liangzhong Lim ◽  
Jianxing Song
Keyword(s):  
Significant Interaction ◽  
Bilayer Membrane ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Large Network ◽  
Sequence Complexity ◽  
Recognition Motif ◽  
Rrm Domain ◽  
In Cells

Abstract526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. FUS belongs to an emerging category of proteins which are capable of forming membraneless organelles in cells via phase separation. On the other hand, eukaryotic cells contain a large network of internal membrane systems. Therefore, it is of fundamental importance to address whether membraneless organelles can interact with membranes. Here we attempted to explore this by NMR HSQC titrations of three FUS domains with gradual addition of DMPC/DHPC bicelle, which mimics the bilayer membrane. We found that both N- and C-terminal LC domains showed no significant interaction with bicelle, but its well-folded RRM domain does dynamically interact with bicelle with an interface opposite to that for binding nucleic acids including RNA and ssDNA. If this in vitro observation also occurs in cells, to interact with membrane might represent a mechanism for dynamically organizing membraneless organelles to membranes to facilitate their physiological functions.

scholarly journals Crystal structure of human Acinus RNA recognition motif domain

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5163 ◽  
Author(s):  
Humberto Fernandes ◽  
Honorata Czapinska ◽  
Katarzyna Grudziaz ◽  
Janusz M. Bujnicki ◽  
Martyna Nowacka
Keyword(s):  
Crystal Structure ◽  
Rna Binding ◽  
Chromatin Condensation ◽  
Rna Recognition Motif ◽  
Target Sequence ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Rrm Domain ◽  
Α Helix ◽  
Β Sheet

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel β-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the β-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.

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