The Transcriptome-wide Landscape and Modalities of EJC Binding in Adult Drosophila

Obrdlík, Aleš; Lin, Gen; Haberman, Nejc; Ule, Jernej; Ephrussi, Anne

doi:10.1016/j.celrep.2019.06.088

Cited by 16 publications

(26 citation statements)

References 98 publications

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“…EJCs bind to mRNAs at specific sites approximately 20–24 nts upstream of the spliced exon-exon junction [ 4 , 5 ]. The observation that this binding is independent of the sequence context and occurs at a specific position relative to the spliced-out intron has puzzled researchers for many years.…”

Section: Spliceosomal Ejc Assemblymentioning

confidence: 99%

“…These revealed that the EJC does not only bind to the canonical deposition site, 20–24 nts upstream of an exon-exon junction, but also at other positions in exons [ 5 , 19 ]. In mammals, these non-canonical binding sites are quite frequent, ranging up to 50% [ 5 ], while in Drosophila nearly all EJCs seem to be deposited at canonical binding sites [ 4 ]. Interestingly, CASC3 containing EJCs also show a strong bias towards canonical deposition, further supporting the assumption that it is not present in all EJC cores [ 44 ].…”

Section: Spliceosomal Ejc Assemblymentioning

confidence: 99%

“…EJCs consist of three invariable core factors ( Figure 1 A) and many interacting proteins, which vary depending on cellular context and gene expression step. The assembly of EJCs during intron splicing results in their binding to mRNAs approximately 20–24 nucleotides (nts) upstream of exon–exon junctions in a sequence-independent manner [ 4 , 5 ]. Due to their remarkably stable interaction with RNA [ 6 , 7 ], EJCs accompany a bound mRNA during its entire lifecycle.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A Day in the Life of the Exon Junction Complex

Schlautmann

Gehring

2020

Biomolecules

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The exon junction complex (EJC) is an abundant messenger ribonucleoprotein (mRNP) component that is assembled during splicing and binds to mRNAs upstream of exon-exon junctions. EJCs accompany the mRNA during its entire life in the nucleus and the cytoplasm and communicate the information about the splicing process and the position of introns. Specifically, the EJC’s core components and its associated proteins regulate different steps of gene expression, including pre-mRNA splicing, mRNA export, translation, and nonsense-mediated mRNA decay (NMD). This review summarizes the most important functions and main protagonists in the life of the EJC. It also provides an overview of the latest findings on the assembly, composition and molecular activities of the EJC and presents them in the chronological order, in which they play a role in the EJC’s life cycle.

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Section: Spliceosomal Ejc Assemblymentioning

confidence: 99%

Section: Spliceosomal Ejc Assemblymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Day in the Life of the Exon Junction Complex

Schlautmann

Gehring

2020

Biomolecules

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“…We find that both Eif4a3 and Magoh, but not a negative control RNA-binding protein HuC, specifically co-immunopreciptate with Rbm8a ( Fig 1A). We and others have previously shown that the EJC primarily binds 24 nts upstream of exon-exon junctions in cultured human cells and adult Drosophila [42][43][44][45][46]. To test if the EJC binds at a similar position on zebrafish spliced RNAs, we first optimized RNA-immunoprecipitation (RIP) from RNase-treated zebrafish embryo lysates using an Rbm8a antibody (S1B and S1C Fig).…”

Section: Introductionmentioning

confidence: 99%

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3′UTR intron-containing NMD targets

et al. 2020

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Many post-transcriptional mechanisms operate via mRNA 3 0 UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination � 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3 0 UTR introns (3 0 UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3 0 UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3 0 UI in zebrafish, mouse and humans. foxo3b is one such proximal 3 0 UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3 0 UIs may serve gene regulatory function in vertebrates.

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“…We have previously shown in human cells that this protein UV-crosslinks to RNA very inefficiently as compared to a sequence-specific RBP HNRNPA1 (Singh et al 2014). Recently, this poor in vivo UV crosslinking ability of EIF4A3 was also reported in Drosophila adult animals (Obrdlik et al 2019). Instead, chemical crosslinking using dithio(bis-) succinimidyl propionate was found to stabilize EIF4A3 within EJCs for identification of Drosophila EJC binding sites.…”

Section: Factors That Impact Uv Crosslinking Ability Of Rna-associatementioning

confidence: 99%

Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA

Patton

Sanjeev

Woodward

et al. 2020

RNA

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In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and-independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.

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The Transcriptome-wide Landscape and Modalities of EJC Binding in Adult Drosophila

Cited by 16 publications

References 98 publications

A Day in the Life of the Exon Junction Complex

A Day in the Life of the Exon Junction Complex

Zebrafish rbm8a and magoh mutants reveal EJC developmental functions and new 3′UTR intron-containing NMD targets

Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA

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