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Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (iCLIP-seq)

ABSTRACT: Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3’ ends and 3’ splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2∆ cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE106477 | GEO | 2019/05/01

REPOSITORIES: GEO

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Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (Structure-seq)

Project description:Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3’ ends and 3’ splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2∆ cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression.

2019-05-01 | GSE106478 | GEO

Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (RNA Pol II ChIP-seq)

Project description:Proper formation of messenger RNA-protein complexes (mRNPs) is critical for accurate gene expression and requires temporal regulation of mRNA structures. RNA helicases are major players that control mRNP/mRNA structures throughout the life of mRNAs. Our group has shown that the DEAD-box RNA helicase Dbp2 in S. cerevisiae is a bona fide RNA helicase in vitro that is required for proper transcription termination and assembly of RNA-binding proteins onto poly(A)+ RNA in vivo. Here, we utilized multiple genome-wide approaches to identify the mRNAs and processing steps targeted by the enzymatic activity of Dbp2. We found that Dbp2 promotes efficient termination of mRNA transcription through remodeling of RNA structures at the 3’ end of mRNAs. This finding elucidates how an RNA helicase regulates gene expression by modulating RNA structures at a genome-wide scale. In addition, Dbp2 facilitates efficient splicing, for which the helicase activity of Dbp2 is required. This reveals a previously uncharacterized role of RNA helicases besides known splicing factors. Overall, our work shows that Dbp2 plays a critical role in pre-mRNA processing and how RNA structure remodeling impacts gene expression.

2019-05-01 | GSE125283 | GEO

RNA sequencing of DDX23 knockdown by siRNA in ovarian cancer cells

Project description:DDX23 belongs to DEAD-box family of RNA helicases and plays crucial roles in spliceosome formation and pre-mRNA splicing. In this study, DDX23 was first identified as a key DEAD-box RNA helicase in ovarian cancer, and its overexpressed was associated with poor clinical outcomes. High expression of DDX23 was involved in the malignant proliferation and aggressiveness of ovarian cancer.

2021-07-31 | GSE181078 | GEO

The RNA helicase CrhR as a regulatory factor

Project description:DEAD-box RNA-helicases catalyze the correct folding of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083) whose inactivation has severe effects on cellular physiology and morphology, particularly under cold stress. In order to identify potential RNA targets of CrhR, custom microarrays containing sequences corresponding to non-coding small RNAs (UTRs, ncRNAs, asRNAs, iRNAs) and all mRNAs were constructed. The RNAs exhibiting CrhR-dependent alteration of expression constitute both mRNA and small RNA transcripts. CrhR therefore is not a global regulator of RNA metabolism but instead interacts with a specific subset of the total RNA repertoire in Synechocystis, a CrhR-specific RNA regulon that functions predominately at low temperature. RNA half-life analyses indicated that expression of regulon members is controlled by a mechanism that does not involve CrhR-dependent RNA turnover. Biochemically, CrhR is able to anneal but not unwind an asRNA-mRNA target in vitro, providing evidence that regulation occurs through an RNA duplex intermediate. The data reveal a novel mechanism regulating the differential expression of operon members, crhR mutation disrupts a negative regulatory feedback loop involving CrhR regulation at an early stage of transcription in affected operons. We propose that CrhR is functioning as a RNA chaperone, similar to Hfq in enterobacteria, but regulating both post-transcriptional events and transcription termination, by altering sRNA metabolism. We monitored gene expression of an Synechocystis PCC6083 WT and a partial crhR knockout at 30M-BM-0C and after 3h of culturing at 20M-BM-0C. Each sample was done in biological triplicates.

2014-06-17 | E-GEOD-58544 | biostudies-arrayexpress

The RNA helicase CrhR as a regulatory factor

2014-06-17 | GSE58544 | GEO

Analysis of RIP-seq of Ded1 and eIF4A(Tif1) with and without glucose depletion

Project description:The aim of this experiment was to look at RNAs which associate with DEAD-box RNA helicases with and without 10 minutes of glucose starvation.

2021-09-30 | E-MTAB-9095 | biostudies-arrayexpress

DEAD box RNA helicases are pervasive protein kinase interactors and activators

Project description:DEAD box (DDX) RNA helicases are a large family of ATPases, many of which have unknown functions. There is emerging evidence that besides their role in RNA biology, DDX proteins may stimulate protein kinases. To investigate if protein kinase-DDX interaction is a more widespread phenomenon, we conducted three orthogonal large-scale screens, including proteomics analysis with 32 RNA helicases, protein array profiling, and kinome-wide in vitro kinase assays. We retrieved Ser/Thr protein kinases as prominent interactors of RNA helicases and report hundreds of binary interactions. We extracted members of eleven protein kinase families, which bind to - and are stimulated by - DDX proteins, including CAMK, CDK, CK1, CK2, DYRK, MARK, NEK, PRKC, SPRK, STE7/MAP2K, and STE20/PAK family members. We identified MARK1 in all screens and validated that DDX proteins accelerate MARK1 catalytic rate. The findings indicate pervasive interactions between protein kinases and DEAD box RNA helicases and provide a rich resource to explore their regulatory relationships.

2024-03-10 | PXD043289 | Pride

Xrn1-sensitive lncRNAs accumulate upon inactivation of Dbp2 and Mtr4

Project description:To investigate the role of the helicases Dbp2 and Mtr4 in the decay of Xrn1-sensitive lncRNAs, we performed RNA-Seq in yeast cells lacking Dbp2 or depleted for Mtr4.

2023-07-27 | GSE235552 | GEO

eIF4A3 interactome by IP-MS in mouse ESCs

Project description:RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNAi screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). We mapped the eIF4A3 interactomes in mouse ESCs, revealing that eIF4A3 is widely involved in the post-transcriptional regulation of gene expression. Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes cellular differentiation via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during cell cycle progression of ESCs. Our results reveal a previously unappreciated role of eIF4A3 and its associated EJC in the post-transcriptional cell cycle control in maintaining stem cell pluripotency.

2023-03-11 | PXD031362 | Pride

Characterization of R-loop-interacting proteins in embryonic stem cells reveals roles in ribosomal RNA processing and gene expression

Project description:Chromatin-associated RNAs have diverse roles in the nucleus. However, their mechanisms of action are poorly understood, in part due to the inability to identify proteins that specifically associate with chromatin-bound RNAs. Here, we address this problem for a subset of chromatin-associated RNAs that form R-loops—RNA-DNA hybrid structures that include a displaced strand of single-stranded DNA. R-loops generally form co-transcriptionally and have important roles in regulation of gene expression, immunoglobulin class switching, and other processes. However, unresolved R-loops can lead to DNA damage and chromosome instability. To identify factors that may bind and potentially regulate R-loop accumulation or mediate R-loop-dependent functions, we used a comparative immunoprecipitation/mass spectrometry approach, with and without RNA-protein crosslinking, to identify a stringent set of R-loop-binding proteins in mouse embryonic stem cells. We identified 365 R-loop-interacting proteins, which were highly enriched for proteins with predicted RNA-binding functions. We characterized several R-loop-interacting proteins of the DEAD-box family of RNA helicases and found that these proteins localize to the nucleolus and, to a lesser degree, the nucleus. Consistent with their localization patterns, we found that these helicases are required for ribosomal RNA processing and regulation of gene expression. Surprisingly, depletion of these helicases resulted in misregulation of highly overlapping sets of protein-coding genes, including many genes that function in differentiation and development. We conclude that R-loop-interacting DEAD-box helicases have non-redundant roles that are critical for maintaining the normal embryonic stem cell transcriptome.

2021-09-17 | PXD022697 | Pride

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