In Vivo Crosslinking of Histone and RNA-Binding Proteins

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.

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

10.1101/287953 ◽

2018 ◽

Cited By ~ 2

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]

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A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5323 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5323-5330 ◽

Cited By ~ 23

Author(s):

S A Amero ◽

M J Matunis ◽

E L Matunis ◽

J W Hockensmith ◽

G Raychaudhuri ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Polytene Chromosomes ◽

Cell Extract ◽

Sequence Motifs ◽

Drosophila Cell ◽

Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3668 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3668-3678 ◽

Cited By ~ 110

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.

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

Biomolecules ◽

10.3390/biom10060872 ◽

2020 ◽

Vol 10 (6) ◽

pp. 872 ◽

Cited By ~ 1

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.

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Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-Binding Proteins in Plants

Genes ◽

10.3390/genes11020207 ◽

2020 ◽

Vol 11 (2) ◽

pp. 207 ◽

Cited By ~ 2

Author(s):

Leandro Lucero ◽

Jeremie Bazin ◽

Johan Rodriguez Melo ◽

Fernando Ibañez ◽

Martín D. Crespi ◽

...

Keyword(s):

Alternative Splicing ◽

Medicago Truncatula ◽

Noncoding Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nodule Development ◽

Nuclear Speckle ◽

Symbiotic Nodule

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicago truncatula, involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40. The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR (ASCO) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicago truncatula and Lotus japonicus, their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40, as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6402 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6402-6409 ◽

Cited By ~ 38

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.

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

Life Science Alliance ◽

10.26508/lsa.201800187 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800187 ◽

Cited By ~ 8

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.

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Screening for New Interaction Partners for a Group IIB Intron Ribozyme with Surface Plasmon Resonance Imaging Coupled to MALDI Mass Spectrometry

10.1101/2020.05.07.082776 ◽

2020 ◽

Author(s):

Ulrike Anders ◽

Maya Gulotti-Georgieva ◽

Susann Zelger-Paulus ◽

Fatima-Ezzahra Hibti ◽

Chiraz Frydman ◽

...

Keyword(s):

Mass Spectrometry ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Living Organism ◽

Maldi Mass Spectrometry ◽

Mitochondrial Fraction ◽

Ionization Mass ◽

Rna Maturation

ABSTRACTRNA maturation is a highly regulated process whose precision is indispensable for the correct transfer of genetic information and, thus, the survival of any living organism. While in higher eukaryotes, this process is known to be assisted by the spliceosome, a very complex system assembled from numerous proteins, in bacteria splicing is catalyzed by ribozymes only. In lower eukaryotes however, e.g., yeast, RNA maturation is also expected to be less complex or simplified. Here, we focus on the mitochondrial group IIB intron RNA Sc.ai5γ from Saccharomyces cerevisiae (Sc.) and Mss116, a protein of the DEAD-box helicase family, known to play a crucial role in the Sc.ai5γ maturation pathway, acting as a co-factor in vivo. Although to date, only Mss116 has been described to be involved in the maturation of Sc.ai5γ, we hypothesize that the folding and splicing of Sc.ai5γ is regulated by more than one protein co-factor, i.e., that a complex or series of several proteins participate in folding and splicing the immature RNA correctly. For the identification of new potential Sc.ai5γ binders we coupled SPR imaging with matrix-assisted laser desorption/ionization mass spectrometry. This combination results in a powerful method to screen for specific RNA-binding proteins from complex mixtures, specifically lysate of the coarse mitochondrial fraction from yeast. Our results indicate that several proteins other than the well-known co-factor Mss116 interact with Sc.ai5γ, namely Dbp8, Prp8, Mrp13, and Cullin-3. With this novel approach, we report the identification of RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach.

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Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

10.1101/321174 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gerard Minuesa ◽

Steven K. Albanese ◽

Arthur Chow ◽

Alexandra Schurer ◽

Sun-Mi Park ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Small Molecule ◽

Myeloid Leukemia ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Wide Spectrum ◽

Gene Expression Signature ◽

Acute Myeloid

SUMMARYThe MUSASHI family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We found that the small molecule Ro 08–2750 (Ro) directly binds to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells resulted in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrated in vivo inhibition of c-MYC and reduced disease burden in a murine AML leukemia model. Thus, we have identified a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

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