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

1998 ◽  
Vol 18 (2) ◽  
pp. 685-693 ◽  
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.

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

Gene ◽  
1997 ◽  
Vol 186 (1) ◽  
pp. 21-27 ◽  
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

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

1999 ◽  
Vol 112 (24) ◽  
pp. 4501-4512 ◽  
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.

scholarly journals RNA recognition motifs of disease-linked RNA-binding proteins contribute to amyloid formation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sashank Agrawal ◽  
Pan-Hsien Kuo ◽  
Lee-Ya Chu ◽  
Bagher Golzarroshan ◽  
Monika Jain ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Amyloid Formation ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

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

1997 ◽  
Vol 17 (5) ◽  
pp. 2649-2657 ◽  
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.

scholarly journals RNA recognition motifs involved in nuclear import of RNA-binding proteins

RNA Biology ◽  
2010 ◽  
Vol 7 (3) ◽  
pp. 339-344 ◽  
Author(s):  
Alejandro Cassola ◽  
Griselda Noé ◽  
Alberto C. Frasch
Keyword(s):  
Nuclear Import ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

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.

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