scholarly journals Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Chase A Weidmann ◽  
Chen Qiu ◽  
René M Arvola ◽  
Tzu-Fang Lou ◽  
Jordan Killingsworth ◽  
...  
Keyword(s):  
Regulatory Network ◽  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Recognition ◽  
Recognition Sequence ◽  
Repression Complex ◽  
Neuronal Functions ◽  
Rna Complexes

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

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 An in vivo binding assay for RNA-binding proteins based on repression of a reporter gene

2017 ◽  
Author(s):  
Noa Katz ◽  
Roni Cohen ◽  
Oz Solomon ◽  
Beate Kaufmann ◽  
Noa Eden ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Translational Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Coat Proteins ◽  
High Throughput Assay

ABSTRACTWe employ a reporter assay and Selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-seq) to study translational regulation by RNA-binding proteins, in bacteria. We designed 82 constructs, each with a single hairpin based on the binding sites of the RNA-binding coat proteins of phages MS2, PP7, GA, and Qβ, at various positions within the N-terminus of a reporter gene. In the absence of RNA-binding proteins, the translation level depends on hairpin location, and exhibits a three-nucleotide periodicity. For hairpin positions within the initiation region, we observe strong translational repression in the presence of its cognate RNA-binding protein. In vivo SHAPE-seq results for a representative construct indicate that the repression phenomenon correlates with a wide-swath of protection, including the hairpin and extending past the ribosome binding site. Consequently, our data suggest that the protection provided by the RBP-hairpin complex inhibits ribosomal initiation. Finally, utilizing the repression phenomenon for quantifying protein-RNA binding affinity in vivo, we both observe partially contrasting results to previous in vitro and in situ studies, and additionally, show that this method can be used in a high-throughput assay for a quantitative study of protein-RNA binding in vivo.

scholarly journals Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

1994 ◽  
Vol 14 (9) ◽  
pp. 5898-5909 ◽  
Author(s):  
R Stripecke ◽  
C C Oliveira ◽  
J E McCarthy ◽  
M W Hentze
Keyword(s):  
Binding Sites ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Yeast Cells ◽  
General Mechanism ◽  
Translational Repressor

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

scholarly journals CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

1998 ◽  
Vol 18 (9) ◽  
pp. 5000-5009 ◽  
Author(s):  
Dong Yan ◽  
Rhonda Perriman ◽  
Haller Igel ◽  
Kenneth J. Howe ◽  
Megan Neville ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Normal Activity ◽  
Binding Activity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Yeast Homolog

ABSTRACT A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells

1994 ◽  
Vol 14 (9) ◽  
pp. 5898-5909
Author(s):  
R Stripecke ◽  
C C Oliveira ◽  
J E McCarthy ◽  
M W Hentze
Keyword(s):  
Binding Sites ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Yeast Cells ◽  
General Mechanism ◽  
Translational Repressor

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

scholarly journals Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

2021 ◽  
Vol 17 (12) ◽  
pp. e1009658
Author(s):  
Valentin Schneider-Lunitz ◽  
Jorge Ruiz-Orera ◽  
Norbert Hubner ◽  
Sebastiaan van Heesch
Keyword(s):  
Translational Regulation ◽  
Binding Proteins ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Translational Efficiency ◽  
Splicing Factors ◽  
Biological Processes ◽  
Differential Affinity

RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8—all identified as multifunctional RBPs by our analysis—selectively influence target translation rates depending on 5’ UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

scholarly journals RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

scholarly journals Architecture of RNA influences plant biology

2021 ◽  
Author(s):  
Jiaying Zhu ◽  
Changhao Li ◽  
Xu Peng ◽  
Xiuren Zhang
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mirna Biogenesis ◽  
Plant Responses ◽  
Tertiary Structures ◽  
Rna Transcripts ◽  
Environmental Variations ◽  
Living Organisms ◽  
Processing Stability

Abstract The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick, Hoogsteen or wobble base pairing. In vivo, RNA folding is not a simple thermodynamics event of minimizing free energy. Instead, the process is constrained by transcription, RNA binding proteins (RBPs), steric factors and micro-environment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid-liquid phase separation and plant responses to environmental variations such as temperature and salinity. At the molecular level, RSS is correlated with regulating splicing, polyadenylation, protein systhsis, and miRNA biogenesis and functions. In this review, we summarized newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology.

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