scholarly journals Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3′ Splice-Site Selection

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1865
Author(s):  
Jana Kralovicova ◽  
Ivana Borovska ◽  
Monika Kubickova ◽  
Peter J. Lukavsky ◽  
Igor Vorechovsky
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Branch Points ◽  
Rna Recognition Motifs ◽  
Small Nuclear Ribonucleoprotein ◽  
Isoform Diversity ◽  
Protein Properties ◽  
Recognition Motifs ◽  
Nuclear Ribonucleoprotein ◽  
Mrna Isoform

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

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 Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H

2018 ◽  
Vol 140 (37) ◽  
pp. 11661-11673 ◽  
Author(s):  
Srinivasa R. Penumutchu ◽  
Liang-Yuan Chiu ◽  
Jennifer L. Meagher ◽  
Alexandar L. Hansen ◽  
Jeanne A. Stuckey ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Recognition Motifs ◽  
Nuclear Ribonucleoprotein

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.

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