scholarly journals Minimal functional domains of paralogues hnRNP L and hnRNP LL exhibit mechanistic differences in exonic splicing repression

2013 ◽  
Vol 453 (2) ◽  
pp. 271-279 ◽  
Author(s):  
Ganesh Shankarling ◽  
Kristen W. Lynch
Keyword(s):  
Sequence Similarity ◽  
Expression Patterns ◽  
Rna Recognition ◽  
High Sequence Similarity ◽  
Rna Recognition Motifs ◽  
Hnrnp Proteins ◽  
Functional Consequences ◽  
Recognition Motifs ◽  
Nuclear Ribonucleoprotein ◽  
Specific Control

Understanding functional distinctions between related splicing regulatory proteins is critical to deciphering tissue-specific control of alternative splicing. The hnRNP (heterogeneous nuclear ribonucleoprotein) L and hnRNP LL (hnRNP L-like) proteins are paralogues that have overlapping, but distinct, expression patterns and functional consequences. These two proteins share high sequence similarity in their RRMs (RNA-recognition motifs), but diverge in regions outside of the RRMs. In the present study, we use an MS2-tethering assay to delineate the minimal domains of hnRNP L and hnRNP LL which are required for repressing exon inclusion. We demonstrate that for both proteins, regions outside the RRMs, the N-terminal region, and a linker sequence between RRMs 2 and 3, are necessary for exon repression, but are only sufficient for repression in the case of hnRNP LL. In addition, both proteins require at least one RRM for maximal repression. Notably, we demonstrate that the region encompassing RRMs 1 and 2 of hnRNP LL imparts a second silencing activity not observed for hnRNP L. This additional functional component of hnRNP LL is consistent with the fact that the full-length hnRNP LL has a greater silencing activity than hnRNP L. Thus the results of the present study provide important insight into the functional and mechanistic variations that can exist between two highly related hnRNP proteins.

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 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 The Coactivator activator CoAA regulates PEA3 group member transcriptional activity

2011 ◽  
Vol 439 (3) ◽  
pp. 469-477 ◽  
Author(s):  
Kathye Verreman ◽  
Jean-Luc Baert ◽  
Alexis Verger ◽  
Hervé Drobecq ◽  
Elisabeth Ferreira ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Human Cancer ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Human Cancer Cells ◽  
Important Regulator ◽  
Group Members ◽  
Recognition Motifs ◽  
Nuclear Ribonucleoprotein ◽  
Group Member

The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.

scholarly journals Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling

2005 ◽  
Vol 25 (1) ◽  
pp. 150-162 ◽  
Author(s):  
Francesca Vitali ◽  
Anke Henning ◽  
Florian C Oberstrass ◽  
Yann Hargous ◽  
Sigrid D Auweter ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Segmental Isotope Labeling ◽  
Recognition Motifs

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.

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