Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE99616 | GEO | 2017/09/28
SECONDARY ACCESSION(S): PRJNA388894
REPOSITORIES: GEO
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Project description:BackgroundFunctions for RNA-binding proteins in orchestrating plant development and environmental responses are well established. However, the lack of a genome-wide view of their in vivo binding targets and binding landscapes represents a gap in understanding the mode of action of plant RNA-binding proteins. Here, we adapt individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) genome-wide to determine the binding repertoire of the circadian clock-regulated Arabidopsis thaliana glycine-rich RNA-binding protein AtGRP7.ResultsiCLIP identifies 858 transcripts with significantly enriched crosslink sites in plants expressing AtGRP7-GFP that are absent in plants expressing an RNA-binding-dead AtGRP7 variant or GFP alone. To independently validate the targets, we performed RNA immunoprecipitation (RIP)-sequencing of AtGRP7-GFP plants subjected to formaldehyde fixation. Of the iCLIP targets, 452 were also identified by RIP-seq and represent a set of high-confidence binders. AtGRP7 can bind to all transcript regions, with a preference for 3' untranslated regions. In the vicinity of crosslink sites, U/C-rich motifs are overrepresented. Cross-referencing the targets against transcriptome changes in AtGRP7 loss-of-function mutants or AtGRP7-overexpressing plants reveals a predominantly negative effect of AtGRP7 on its targets. In particular, elevated AtGRP7 levels lead to damping of circadian oscillations of transcripts, including DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN2 and CCR-LIKE. Furthermore, several targets show changes in alternative splicing or polyadenylation in response to altered AtGRP7 levels.ConclusionsWe have established iCLIP for plants to identify target transcripts of the RNA-binding protein AtGRP7. This paves the way to investigate the dynamics of posttranscriptional networks in response to exogenous and endogenous cues.
| S-EPMC5663106 | biostudies-literature
Project description:Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7
| PRJNA388894 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-01-11 | GSE223862 | GEO
Project description:Protein recognition and binding, which result in either transient or long-lived complexes, play a fundamental role in many biological functions, but sometimes also result in pathologic aggregates. We use a simplified simulation model to survey a range of systems where two highly flexible protein chains form a homodimer. In all cases, this model, which corresponds to a perfectly funneled energy landscape for folding and binding, reproduces the macroscopic experimental observations on whether folding and binding are coupled in one step or whether intermediates occur. Owing to the minimal frustration principle, we find that, as in the case of protein folding, the native topology is the major factor that governs the choice of binding mechanism. Even when the monomer is stable on its own, binding sometimes occurs fastest through unfolded intermediates, thus showing the speedup envisioned in the fly-casting scenario for molecular recognition.
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Project description:Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.
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Project description:The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7?-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7?-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-10-29 | E-GEOD-72720 | biostudies-arrayexpress
Project description:About half of all human and mouse miRNA genes are located within introns of protein-coding genes. Despite this, little is known about functional interactions between miRNAs and their host genes. The intronic miRNA miR-128 regulates neuronal excitability and controls dendrite outgrowth of projection neurons during development of the mouse cerebral cortex. Its host genes R3hdm1 and Arpp21 encode highly conserved, putative RNA-binding proteins. Here we use iCLIP to describe the RNA-binding activity of ARPP21, which recognizes uridine-rich sequences with exquisite sensitivity for 3UTRs. Surprisingly, ARPP21 antagonizes miR-128 activity by co-regulating a subset of miR-128 target mRNAs enriched for neurodevelopmental functions. In contrast to miR-128, we show that ARPP21 acts as a positive post-transcriptional regulator, at least in part through interaction with the eukaryotic translation initiation complex eIF4F. This molecular antagonism is also reflected in inverse activities during dendritogenesis: miR-128 overexpression or knockdown of ARPP21 reduces dendritic complexity; ectopic ARPP21 leads to an increase. The regulatory interaction between ARPP21 and miR-128 is a unique example of convergent function by two products of a single gene.
2018-02-23 | E-MTAB-5911 | biostudies-arrayexpress
Project description:RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA-protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins-SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation-interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA-protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs.
| S-EPMC5737842 | biostudies-literature
Project description:Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5'end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3'end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.
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