A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

Abstract The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

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An essential RNA-binding lysine residue in the Nab3 RRM domain undergoes mono and trimethylation

10.1101/592923 ◽

2019 ◽

Author(s):

Kwan Yin Lee ◽

Anand Chopra ◽

Kyle Biggar ◽

Marc D. Meneghini

Keyword(s):

Rna Binding ◽

Growth Defect ◽

Rna Sequences ◽

Rna Recognition Motifs ◽

Rrm Domain ◽

Lysine Residues ◽

Recognition Motifs ◽

Target Rna

AbstractThe Nrd1-Nab3-Sen1 (NNS) complex integrates molecular inputs to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by methylation of histone H3 lysine-4 as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3, and Sen1 that are mono-, di-, or trimethylated, suggesting novel molecular inputs for NNS regulation. One of these methylated residues, Nab3 lysine-363 (K363), resides within its RRM, and is known to physically contact target RNA. Although mutation of Nab3-K363 to arginine (Nab3-K363R) causes a severe growth defect, it nevertheless produces a stable protein that is incorporated into the NNS complex, suggesting that RNA binding through Nab3-K363 is crucial for NNS function. Consistent with this hypothesis, K363R mutation decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects in vivo. These findings reveal crucial roles for Nab3-K363 and suggest that methylation of this residue may modulate NNS activity through its impact on Nab3 RNA binding.

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A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2649 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2649-2657 ◽

Cited By ~ 49

Author(s):

H Shi ◽

B E Hoffman ◽

J T Lis

Keyword(s):

Rna Binding ◽

Rna Recognition ◽

Loop Structure ◽

Hairpin Loop ◽

Structural Element ◽

Rna Sequences ◽

Sr Protein ◽

Rna Recognition Motifs ◽

Splicing Regulators ◽

Recognition Motifs

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

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Bioassaying Putative RNA-Binding Motifs in a Protein Encoded by a Gene That Influences Courtship and Visually Mediated Behavior in Drosophila: In Vitro Mutagenesis of nonA

Genetics ◽

10.1093/genetics/143.1.259 ◽

1996 ◽

Vol 143 (1) ◽

pp. 259-275

Author(s):

Ralf Stanewsky ◽

Thomas A Fry ◽

Ingolf Reim ◽

Harald Saumwebert ◽

Jeffrey C Hall

Keyword(s):

Rna Binding ◽

Courtship Song ◽

In Vitro Mutagenesis ◽

Rna Recognition ◽

Null Mutant ◽

Rna Recognition Motifs ◽

Binding Motifs ◽

Recognition Motifs ◽

Transgenic Strains

Abstract The no-on-transient-A (nonA) gene of Drosophila melanogaster influences vision, courtship song, and viability. The nod-encoded polypeptide is inferred to bind single-stranded nucleic acids. Although sequence-analysis of NONA implies that it belongs to a special interspecific family of this protein type, it does contain two classical RNA recognition motifs (RRM). Their behavioral significance was assayed by generating transgenic strains that were singly or multiply mutated within the relatively N-terminal motif (RRM1) or within RRM2. Neither class of mutation affected NONA binding to polytene chromcsomes. The former mutations led to extremely low viability, accompanied by diminished adult longevities that were much worse than for a nod-null mutant, implying that faulty interpolypeptide interactions might accompany the effects of the amino-acid substitutions within RRM1. All in vitro-mutated types caused optomotor blindness and an absence of transient spikes in the electroretinogram. Courtship analysis discriminated between the effects of the mutations: the RRM2-mutated type generated song pulses and trains that tended to be mildly mutant. These phenotypic abnormalities reinforce the notion that nonA' s ubiquitous expression has its most important consequences in the optic lobes, the thoracic ganglia, or both, depending in part on the nonA allele.

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NMR Fragment-Based Screening against Tandem RNA Recognition Motifs of TDP-43

International Journal of Molecular Sciences ◽

10.3390/ijms20133230 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3230 ◽

Cited By ~ 4

Author(s):

Gilbert Nshogoza ◽

Yaqian Liu ◽

Jia Gao ◽

Mingqing Liu ◽

Sayed Ala Moududee ◽

...

Keyword(s):

Protein Interactions ◽

Nuclear Protein ◽

Rna Binding ◽

Pathological Condition ◽

Rna Recognition ◽

Protein Protein Interactions ◽

Rna Recognition Motifs ◽

Rrm Domain ◽

Recognition Motifs ◽

Lead Evolution

The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the 15N-labeled tandem RRM and RRM2, respectively, and modeled by the CSP-guided High Ambiguity Driven biomolecular DOCKing (HADDOCK). These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.

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Faculty Opinions recommendation of The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025155.298750 ◽

2005 ◽

Author(s):

Reinhard Lührmann

Keyword(s):

Rna Binding ◽

Splicing Factor ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Recognition Motifs ◽

U6 Rna

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An RNA-binding protein gene (RBP1) of Saccharomyces cerevisiae encodes a putative glucose-repressible protein containing two RNA recognition motifs

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)82440-1 ◽

1993 ◽

Vol 268 (20) ◽

pp. 15080-15087

Author(s):

F.J. Lee ◽

J. Moss

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Binding ◽

Protein Gene ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Binding Protein Gene ◽

Recognition Motifs

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Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.685 ◽

1998 ◽

Vol 18 (2) ◽

pp. 685-693 ◽

Cited By ~ 108

Author(s):

Laura E. Hake ◽

Raul Mendez ◽

Joel D. Richter

Keyword(s):

Zinc Finger ◽

Rna Binding ◽

Rna Recognition ◽

Cytoplasmic Polyadenylation ◽

Functional Motif ◽

Rna Recognition Motifs ◽

Amino Terminal ◽

Terminal Amino ◽

Maternal Mrnas ◽

Recognition Motifs

ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

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Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1

Gene ◽

10.1016/s0378-1119(96)00673-7 ◽

1997 ◽

Vol 186 (1) ◽

pp. 21-27 ◽

Cited By ~ 14

Author(s):

Yasuyuki Kurihara ◽

Takashi Nagata ◽

Takao Imai ◽

Ado Hiwatashi ◽

Masataka Horiuchi ◽

...

Keyword(s):

Structural Properties ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Recognition Motifs

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Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

Journal of Cell Science ◽

10.1242/jcs.112.24.4501 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4501-4512 ◽

Cited By ~ 1

Author(s):

Y.M. Yannoni ◽

K. White

Keyword(s):

Nuclear Localization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Localization Sequence ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Mutant Proteins ◽

Localization Sequence ◽

Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

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