scholarly journals mRNA structure dynamics identifies the embryonic RNA regulome

2018 ◽  
Author(s):  
Jean-Denis Beaudoin ◽  
Eva Maria Novoa ◽  
Charles E Vejnar ◽  
Valeria Yartseva ◽  
Carter Takacs ◽  
...  
Keyword(s):  
Rna Structure ◽  
Regulatory Elements ◽  
Structure Dynamics ◽  
Mrna Structure ◽  
Maternal To Zygotic Transition ◽  
Gene Regulatory ◽  
Folded Structures ◽  
Affect Gene Expression

RNA folding plays a crucial role in RNA function. However, our knowledge of the global structure of the transcriptome is limited to steady-state conditions, hindering our understanding of how RNA structure dynamics influences gene function. Here, we have characterized mRNA structure dynamics during zebrafish development. We observe that on a global level, translation guides structure rather than structure guiding translation. We detect a decrease in structure in translated regions, and we identify the ribosome as a major remodeler of RNA structure in vivo. In contrast, we find that 3’-UTRs form highly folded structures in vivo, which can affect gene expression by modulating miRNA activity. Furthermore, we find that dynamic 3’-UTR structures encode RNA decay elements, including regulatory elements in nanog and cyclin A1, key maternal factors orchestrating the maternal-to-zygotic transition. These results reveal a central role of RNA structure dynamics in gene regulatory programs.

scholarly journals Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

2019 ◽  
Author(s):  
Minglei Yang ◽  
Hugh C. Woolfenden ◽  
Yueying Zhang ◽  
Xiaofeng Fang ◽  
Qi Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Target Site ◽  
Site Structure ◽  
Mrna Structure ◽  
Sequence Complementarity ◽  
Primary Determinant ◽  
Site Binding

ABSTRACTMicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-Induced Silencing Complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. Here, we investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, the unfolding of the target site structure is the primary determinant for miRISC activity in vivo. Notably, we found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent structure Motif (TAM), can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can regulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

scholarly journals Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo

2020 ◽  
Vol 48 (15) ◽  
pp. 8767-8781 ◽  
Author(s):  
Minglei Yang ◽  
Hugh C Woolfenden ◽  
Yueying Zhang ◽  
Xiaofeng Fang ◽  
Qi Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Target Site ◽  
Site Structure ◽  
Mrna Structure ◽  
In Silico Studies ◽  
Sequence Complementarity ◽  
Site Binding

Abstract MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

scholarly journals Quantitative prediction of variant effects on alternative splicing using endogenous pre-messenger RNA structure probing

2021 ◽  
Author(s):  
Jayashree Kumar ◽  
Lela Lackey ◽  
Justin M. Waldern ◽  
Abhishek Dey ◽  
David H. Mathews ◽  
...  
Keyword(s):  
Rna Structure ◽  
Messenger Rna ◽  
Regulatory Elements ◽  
Quantitative Prediction ◽  
Mrna Structure ◽  
Splicing Regulatory Elements ◽  
Alternatively Spliced ◽  
Endogenous Precursor ◽  
Precursor Mrna

AbstractSplicing is a highly regulated process that depends on numerous factors. It is particularly challenging to quantitatively predict how a mutation will affect precursor messenger RNA (mRNA) structure and the subsequent functional consequences. Here we use a novel Mutational Profiling (-MaP) methodology to obtain highly reproducible endogenous precursor and mature mRNA structural probing data in vivo. We use these data to estimate Boltzmann suboptimal ensembles, and predict the structural consequences of mutations on precursor mRNA structure. Together with a structural analysis of recent cryo-EM spliceosome structures at different stages of the splicing cycle, we determined that the footprint of the Bact complex on precursor mRNA is best able to predict splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene. However, structure alone only achieves 74% accuracy. We therefore developed a β-regression weighting framework that incorporates splice site strength, structure and exonic/intronic splicing regulatory elements which together achieves 90% accuracy for 47 known and six newly discovered splice-altering variants. This combined experimental/computational framework represents a path forward for accurate prediction of splicing related disease-causing variants.

scholarly journals Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

1989 ◽  
Vol 9 (12) ◽  
pp. 5315-5323 ◽  
Author(s):  
J Imbert ◽  
M Zafarullah ◽  
V C Culotta ◽  
L Gedamu ◽  
D Hamer
Keyword(s):  
Gene Transcription ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Direct Role ◽  
Transcription In Vitro ◽  
Mouse Cells ◽  
Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

scholarly journals Targeted Disruption of the Basic Krüppel-Like Factor Gene (Klf3) Reveals a Role in Adipogenesis

2008 ◽  
Vol 28 (12) ◽  
pp. 3967-3978 ◽  
Author(s):  
Nancy Sue ◽  
Briony H. A. Jack ◽  
Sally A. Eaton ◽  
Richard C. M. Pearson ◽  
Alister P. W. Funnell ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Adipocyte Differentiation ◽  
Regulatory Elements ◽  
Targeted Disruption ◽  
Embryonic Fibroblasts ◽  
Factor Gene ◽  
Gene Regulatory Elements ◽  
Gene Regulatory ◽  
Fat Pads

ABSTRACT Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpα promoter. We find that C/ebpα is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpα promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.

scholarly journals CNBP controls transcription by unfolding DNA G-quadruplex structures

2019 ◽  
Vol 47 (15) ◽  
pp. 7901-7913 ◽  
Author(s):  
Aldana P David ◽  
Angélique Pipier ◽  
Federico Pascutti ◽  
Andrés Binolfi ◽  
Andrea M J Weiner ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Regulatory Elements ◽  
Transcription Start Sites ◽  
G4 Dna ◽  
Dna Strands ◽  
Living Organisms ◽  
And Function

Abstract Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG4T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN (NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.

The Genetics of Evolutionary Novelties

2014 ◽  
Author(s):  
Günter P. Wagner
Keyword(s):  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Regulatory Elements ◽  
Complex Problem ◽  
Micro Rnas ◽  
Sex Comb ◽  
Gene Regulatory ◽  
Transcription Factor Proteins ◽  
Evolutionary Novelties

This chapter examines the molecular genetics of evolutionary novelties. In particular, it investigates which molecular mechanisms might be involved in the origination of novel gene regulatory networks (and, thus, character identity networks) and what these mechanisms imply for the origin of novel characters. The chapter begins with a discussion of the complex problem of the evolution of transcriptional regulation by focusing on the evolution of cis-regulatory elements (CREs) and the evolution of transcription factor proteins. It then asks whether novel pigment spots, such as the Drosophila wing spots, are novelties. It also explores an evolutionary novelty known as sex comb and the role of transposable elements in the origin of novel CREs. Finally, it considers the role of gene duplications, the evolution of micro-RNAs (miRNAs), and the possibility of a mechanistic difference between adaptation and innovation.

scholarly journals Functional Interaction of CP2 with GATA-1 in the Regulation of Erythroid Promoters

2006 ◽  
Vol 26 (10) ◽  
pp. 3942-3954 ◽  
Author(s):  
Francesca Bosè ◽  
Cristina Fugazza ◽  
Maura Casalgrandi ◽  
Alessia Capelli ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Physical Interaction ◽  
Regulatory Regions ◽  
Dna Mutations ◽  
Genes Encoding ◽  
A Site

ABSTRACT We observed that binding sites for the ubiquitously expressed transcription factor CP2 were present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), we demonstrated the functional importance of the adjacent CP2/GATA-1 sites. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impaired HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 were present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes.

scholarly journals Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Marat Sabirov ◽  
Olga Kyrchanova ◽  
Galina V. Pokholkova ◽  
Artem Bonchuk ◽  
Natalia Klimenko ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Open Chromatin ◽  
Wild Type ◽  
Long Distance ◽  
Regulatory Domains ◽  
Time Interaction ◽  
Architectural Proteins

AbstractBackgroundPita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of threehoxgenes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins.ResultsUsing in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of thewild-typeand mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions.ConclusionsThe results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

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