Dataset Information

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress.

ABSTRACT: RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (?CRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein-RNA interactions in vivo on a minute time-scale. Here, using ?CRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein-RNA interactions within 1?min following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. ?CRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein-RNA interactions.Protein RNA interactions are dynamic and regulated in response to environmental changes. Here the authors describe 'kinetic CRAC', an approach that allows time resolved analyses of protein RNA interactions with minute time point resolution and apply it to gain insight into the function of the RNA-binding protein Nab3.

SUBMITTER: van Nues R

PROVIDER: S-EPMC5432031 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress.

van Nues Rob R Schweikert Gabriele G de Leau Erica E Selega Alina A Langford Andrew A Franklin Ryan R Iosub Ira I Wadsworth Peter P Sanguinetti Guido G Granneman Sander S

Nature communications 20170411 1

RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (χCRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein-RNA interactions in vivo on a minute time-scale. Here, using χCRAC we measure the global RNA-bindi ...[more]

PMID: 28400552

Similar Datasets

Project description:Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target [1]. In relation to this previous research article, here we expand our observations on the effect of the circular RNA decoy on NNS termination. We aimed at verifying that the Nab3 binding sequence present on the decoy is indeed efficiently sequestering Nab3 as intended by design, leading to the expected decrease of Nab3 binding on NNS targets. We employed the crosslinking and cDNA analysis protocol (CRAC) on yeast cells expressing the circular ncRNA decoy or a control construct. We present data from high-resolution genomewide RNA binding of Nab3 in three independent biological replicates of these S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.

Project description:BackgroundOilseed rape is an excellent candidate for phytoremediation of cadmium (Cd) contaminated soils given its advantages of high biomass, fast growth, moderate metal accumulation, ease of harvesting, and metal tolerance, but the cadmium response pathways in this species (Brassica napus) have yet to be fully elucidated. A combined analysis of miRNA and mRNA expression to infer Cd-induced regulation has not been reported in B. napus.ResultsWe characterized concurrent changes in miRNA and mRNA profiles in the roots and shoots of B. napus seedlings after 10 days of 10 mg/L Cd2+ treatment. Cd treatment significantly affected the expression of 22 miRNAs belonging to 11 families in the root and 29 miRNAs belonging to 14 miRNA families in the shoot. Five miRNA families (MIR395, MIR397, MIR398, MIR408 and MIR858) and three novel miRNAs were differentially expressed in both tissues. A total of 399 differentially expressed genes (DEGs) in the root and 389 DEGs in the shoot were identified, with very little overlap between tissue types. Eight anti-regulation miRNA-mRNA interaction pairs in the root and eight in the shoot were identified in response to Cd and were involved in key plant stress response pathways: for example, four genes targeted by miR398 were involved in a pathway for detoxification of superoxide radicals. Cd stress significantly impacted the photosynthetic pathway. Transcription factor activation, antioxidant response pathways and secondary metabolic processes such as glutathione (GSH) and phenylpropanoid metabolism were identified as major components for Cd-induced response in both roots and shoots.ConclusionsCombined miRNA and mRNA profiling revealed miRNAs, genes and pathways involved in Cd response which are potentially critical for adaptation to Cd stress in B. napus. Close crosstalk between several Cd-induced miRNAs and mRNAs was identified, shedding light on possible mechanisms for response to Cd stress in underground and aboveground tissues in B. napus. The pathways, genes, and miRNAs identified here will be valuable targets for future improvement of cadmium tolerance in B. napus.

Dataset Information

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress.

Publications

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets