scholarly journals Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

2021 ◽  
Author(s):  
Simon H. Stitzinger ◽  
Salma Sohrabi-Jahromi ◽  
Johannes Söding
Keyword(s):  
High Throughput ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Binding Domains

AbstractNumerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA binding domains display low specificity and affinity, to the extent that for most RNA-binding domains, the enrichment of the best binding motif measured by high-throughput RNA SELEX or RNA bind-n-seq is usually below 10-fold, dramatically lower than that of DNA-binding domains. Here, we develop a thermodynamic model to predict the binding affinity for proteins with any number of RNA-binding domains given the affinities of their isolated domains. For the four proteins in which affinities for individual domains have been measured the model predictions are in good agreement with experimental values. The model gives insight into how proteins with multiple RNA-binding domains can reach affinities and specificities orders of magnitude higher than their individual domains. Our results contribute towards resolving the conundrum of missing specificity and affinity of RNA binding proteins and underscore the need for bioinformatic methods that can learn models for multi-domain RNA binding proteins from high-throughput in-vitro and in-vivo experiments.

scholarly journals Validation and classification of RNA binding proteins identified by mRNA interactome capture

2021 ◽  
Author(s):  
Vaishali ◽  
Lyudmila Dimitrova-Paternoga ◽  
Kevin Haubrich ◽  
Mai Sun ◽  
Anne Ephrussi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Modular Organization ◽  
Binding Domains ◽  
Mammalian Cell Lines ◽  
Rna Immunoprecipitation ◽  
Rna Binding Domains ◽  
Rna Binders

AbstractRNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1’s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

scholarly journals PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

2018 ◽  
Author(s):  
Yongjia Duan ◽  
Aiying Du ◽  
Jinge Gu ◽  
Gang Duan ◽  
Chen Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Posttranslational Modifications ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Hnrnp A1 ◽  
Rnp Granules

SUMMARYMutations in RNA-binding proteins localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregations, which are pathological hallmarks in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of PARylation, an important PTM involved in DNA damage repair and cell death, in RNP-related neurodegeneration. We reveal that PARylation levels are a major regulator of the dynamic assembly-disassembly of RNP granules, and the disease-related RNPs such as hnRNP A1 and TDP-43 can both be PARylated and bind to PARylated proteins. We further identify the PARylation site of hnRNP A1 at K298, which controls the cytoplasmic translocation of hnRNP A1 in response to stress, as well as the PAR-binding motif (PBM) of hnRNP A1, which is required for the delivery and association of hnRNP A1 to stress granules. Moreover, we show that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, we establish that both genetic and pharmacological inhibition of PARP mitigates hnRNP A1 and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings indicate a novel and crucial role of PARylation in regulating the assembly and the dynamics of RNP granules, and dysregulation of PARylation may contribute to ALS disease pathogenesis.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

scholarly journals SSMART: Sequence-structure motif identification for RNA-binding proteins

2018 ◽  
Author(s):  
Alina Munteanu ◽  
Neelanjan Mukherjee ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Motif ◽  
Primary Sequence ◽  
Sequence Structure ◽  
Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

scholarly journals A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

1996 ◽  
Vol 16 (7) ◽  
pp. 3668-3678 ◽  
Author(s):  
M F Henry ◽  
P A Silver
Keyword(s):  
Normal Cell ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Temperature Sensitive ◽  
Arginine Residues ◽  
Cognate Gene ◽  
Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.

scholarly journals Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 872 ◽  
Author(s):  
Clemens Grimm ◽  
Jann-Patrick Pelz ◽  
Cornelius Schneider ◽  
Katrin Schäffler ◽  
Utz Fischer
Keyword(s):  
Crystal Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Turnover Rates ◽  
Mrna Transcription ◽  
Terminal Part ◽  
Protein Output

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

scholarly journals The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

1997 ◽  
Vol 17 (11) ◽  
pp. 6402-6409 ◽  
Author(s):  
L Wu ◽  
P J Good ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

scholarly journals A nuclear localization domain in the hnRNP A1 protein.

1995 ◽  
Vol 129 (3) ◽  
pp. 551-560 ◽  
Author(s):  
H Siomi ◽  
G Dreyfuss
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Motif ◽  
Hnrnp A1 ◽  
Binding Domains ◽  
Rich Domain ◽  
Localization Domain ◽  
Hnrnp Proteins

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.

scholarly journals The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity

2018 ◽  
Vol 1 (5) ◽  
pp. e201800187 ◽  
Author(s):  
Daniela Lazzaretti ◽  
Lina Bandholz-Cajamarca ◽  
Christiane Emmerich ◽  
Kristina Schaaf ◽  
Claire Basquin ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Information ◽  
Target Selection ◽  
Mrna Localization ◽  
In Vitro Binding ◽  
Vitro Binding

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar–phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

scholarly journals Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless

2018 ◽  
Author(s):  
Pravin Kumar Ankush Jagtap ◽  
Marisa Müller ◽  
Pawel Masiewicz ◽  
Sören von Bülow ◽  
Nele Merret Hollmann ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Motifs ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Dsrna Binding ◽  
Rna Binding Domains ◽  
Human Orthologue ◽  
Rox Rna

ABSTRACTMaleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

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