Hel-N1/Hel-N2 proteins are bound to poly(A)+ mRNA in granular RNP structures and are implicated in neuronal differentiation

1996 ◽  
Vol 109 (3) ◽  
pp. 579-589 ◽  
Author(s):  
F.B. Gao ◽  
J.D. Keene
Keyword(s):  
Neuronal Differentiation ◽  
Protein Family ◽  
Rna Recognition Motifs ◽  
Mrna Metabolism ◽  
Cell Extracts ◽  
Multiple Protein ◽  
Human Proteins ◽  
Mature Neurons ◽  
Recognition Motifs

Human proteins Hel-N1 and Hel-N2 contain three RNA recognition motifs (RRMs), and are members of a family of proteins highly homologous to Drosophila ELAV, which is essential for neuronal differentiation. Both proteins bind to A+U-rich 3′ untranslated regions of a variety of growth-related mRNAs in vitro. Here we demonstrate that in medulloblastoma cells derived from childhood brain tumors, Hel-N1 and Hel-N2 are mainly expressed in the cytoplasm, but are detectable in the nucleus. Both proteins are associated with polysomes and can be UV-crosslinked to poly(A)+ mRNA in cell extracts. In the cytoplasm the Hel-N1 protein family resides in granular structures that may contain multiple protein molecules bound to each mRNA. Evidence supporting this multimeric ribonucleoprotein (RNP) model includes in vitro reconstitution and competition experiments in which addition of a single RRM (RRM3) can alter complex formation. As in medulloblastoma cells, the Hel-N1 protein family is present in granular particles in the soma and the proximal regions of dendrites of cultured neurons, and colocalizes with ribosomes. In addition, we demonstrate that expression of the Hel-N1 protein family is up-regulated during neuronal differentiation of embryonic carcinoma P19 cells. Our data suggest that the Hel-N1 protein family is associated with the translational apparatus and implicated in both mRNA metabolism and neuronal differentiation. Furthermore, our findings open the possibility that these proteins participate in mRNA homeostasis in the dendrites and soma of mature neurons.

scholarly journals Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

2001 ◽  
Vol 155 (5) ◽  
pp. 775-786 ◽  
Author(s):  
Stefan Hüttelmaier ◽  
Susanne Illenberger ◽  
Irina Grosheva ◽  
Manfred Rüdiger ◽  
Robert H. Singer ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Splicing ◽  
Sequence Motifs ◽  
Rna Recognition Motifs ◽  
Adhesion Sites ◽  
Polypyrimidine Tract Binding Protein ◽  
Associated Proteins ◽  
Recognition Motifs ◽  
Attachment Proteins

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

RNA-recognition motifs and glycine and arginine-rich region cooperatively regulate the nucleolar localization of nucleolin

2020 ◽  
Author(s):  
Mitsuru Okuwaki ◽  
Ai Saotome-Nakamura ◽  
Masashi Yoshimura ◽  
Shoko Saito ◽  
Hiroko Hirawake-Mogi ◽  
...  
Keyword(s):  
Rna Binding ◽  
Point Mutations ◽  
Binding Activity ◽  
Nucleolar Localization ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Mutant Proteins ◽  
Cell Extracts ◽  
Nucleolar Proteins ◽  
Recognition Motifs

Abstract Nucleolin (NCL) is a nucleolar protein that is involved in the regulation of the nucleolar structure and functions, and consists of three distinct regions: the N-terminal region; the middle region, which contains four RNA-recognition motifs (RRMs); and the C-terminal glycine and arginine-rich (GAR) region. The primary function of the RRMs and GAR is thought to be specific RNA binding. However, it is not well understood how these RNA-binding regions of NCL separately or cooperatively regulate its nucleolar localization and functions. To address this issue, we constructed mutant proteins carrying point mutations at the four RRMs individually or deletion of the C-terminal GAR region. We found that the GAR deletion and the mutations in the fourth RRM (RRM4) decreased the nucleolar localization of NCL. Biochemical analyses showed that NCL interacted directly with ribosomal RNAs (rRNAs) and G-rich oligonucleotides, and that this interaction was decreased by mutations at RRM1 and RRM4 and GAR deletion. Although GAR deletion decreased the rRNA-binding activity of NCL, the mutant was efficiently co-precipitated with rRNAs and nucleolar proteins from cell extracts. These contradictory results suggest that NCL stably localizes to the nucleoli via the interactions with rRNAs and nucleolar proteins via GAR, RRM1, and RRM4.

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

2020 ◽  
Vol 48 (6) ◽  
pp. 2897-2911 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Giovanni L Burke ◽  
Ziyan Chen ◽  
Jack F Greenblatt ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Nascent Transcripts ◽  
Target Rna

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.

scholarly journals The intracisternal A-particle proximal enhancer-binding protein activates transcription and is identical to the RNA- and DNA-binding protein p54nrb/NonO.

1997 ◽  
Vol 17 (2) ◽  
pp. 677-686 ◽  
Author(s):  
A Basu ◽  
B Dong ◽  
A R Krainer ◽  
C C Howe
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Peptide Sequence ◽  
Transcription Activator ◽  
Long Terminal Repeats ◽  
F9 Cells ◽  
Cell Extracts ◽  
Recognition Motifs ◽  
Close Sequence

The long terminal repeats of murine intracisternal A particles (IAPs) contain an IAP proximal enhancer (IPE) element that is inactive in murine F9 embryonal carcinoma cells and active in the parietal endoderm cell line PYS-2. The element binds efficiently to a 60-kDa IPE-binding protein (IPEB) present in PYS-2 cells but poorly to F9 proteins, suggesting a role for IPEB in regulating IAP expression. We have purified calf thymus IPEB, which binds to the IPE and transactivates a reporter gene in HeLa cell extracts. Based on the peptide sequence of the purified calf IPEB, we have cloned a 420-bp cDNA and showed that the encoded protein is the homolog of human p54nrb and mouse NonO, which are characterized by the presence of two RNA recognition motifs. We show that p54nrb is an IPE-binding transcription activator with its DNA-binding and activation domains in the N- and C-terminal halves, respectively. The activation domain of p54nrb is active in HeLa, PYS-2, and F9 cells, whereas p54nrb as a whole molecule is active in HeLa and PYS-2 cells but not in F9 cells. Thus, the lack of activity of p54nrb in F9 cells is due to an ineffective DNA-binding domain. We demonstrate that p54nrb also binds to a pre-mRNA. Based on the close sequence relatedness of this protein to PSF, which is required for pre-mRNA splicing in vitro, we discuss the possibility that p54nrb has dual roles in transcription and splicing.

scholarly journals An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

1997 ◽  
Vol 8 (1) ◽  
pp. 97-108 ◽  
Author(s):  
K E McGrath ◽  
J F Smothers ◽  
C A Dadd ◽  
M T Madireddi ◽  
M A Gorovsky ◽  
...  
Keyword(s):  
Casein Kinase Ii ◽  
Ribosomal Gene ◽  
Casein Kinase ◽  
Electron Microscopic ◽  
Rna Recognition Motifs ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Recognition Motifs

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

scholarly journals 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 ◽  
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.

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

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.

scholarly journals RNA-dependent chromatin association of transcription elongation factors and Pol II CTD kinases

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sofia Battaglia ◽  
Michael Lidschreiber ◽  
Carlo Baejen ◽  
Phillipp Torkler ◽  
Seychelle M Vos ◽  
...  
Keyword(s):  
Rna Recognition ◽  
Elongation Factors ◽  
Elongation Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rna Recognition Motifs ◽  
Pol Ii ◽  
Recognition Motifs ◽  
Rna Interaction

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex.

scholarly journals Nuclear Export and Retention Signals in the RS Domain of SR Proteins

2002 ◽  
Vol 22 (19) ◽  
pp. 6871-6882 ◽  
Author(s):  
Demian Cazalla ◽  
Jun Zhu ◽  
Lisa Manche ◽  
Elisabeth Huber ◽  
Adrian R. Krainer ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Sr Proteins ◽  
Nucleocytoplasmic Shuttling ◽  
Sr Protein ◽  
Nuclear Retention ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

ABSTRACT Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and—in certain situations—nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.

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