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Exon Architecture Controls mRNA m6A Suppression and Gene Expression

ABSTRACT: N6–methyladenosine (m6A) is the most abundant mRNA modification and plays crucial roles in diverse physiological processes. Utilizing a Massively Parallel Assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by “suppressors” that prevent m6A deposition in unmethylated transcriptome regions. We identify Exon Junction Complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons, but not in long internal and terminal exons. EJC-suppressed methylation sites co-localize with EJC-suppressed splice sites, suggesting that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162199 | GEO | 2023/02/01

REPOSITORIES: GEO

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Exon junction complex shapes m6A epitranscriptome during splicing

Project description:N6-methyladenosine (m6A) is the most abundant post-transcriptional methylation of mature mRNA and plays a crucial regulatory role in various biological functions, involving development and cancer progression. m6A is deposited by methyltransferase complex composed of METTL3 and METTL14 cotranscriptionally, and removed by demethylase FTO or ALKBH5. m6A is highly enriched within the 3’ UTRs and in the vicinity of the stop codon of mature mRNA. However, the mechanism that causes this distribution pattern is still enigmatic. Here, we show that EJC packages mRNAs to shape mRNA m6A landscape. We first tested the possibility that demethylase removes m6A during spicing and found that ALKBH5 depletion had a minor effect on m6A levels. Thus, we ruled out the demethylation model. We then hypothesized that splicing factors inhibit the methylation process of internal exons, but not the 3’ UTR. Knockdown of EIF4A3, a core component of the exon junction complex, significantly increased m6A levels in mature mRNAs. The hypermethylated sites are enriched in short internal exons. Furthermore, EIF4A3 depletion upregulates METTL3 binding affinity to mRNA to deposit m6As. In conclusion, Our results demonstrate that EIF4A3 blocks METTL3 binding and methylating internal short exons of mRNA. These findings shed light on the fact that RNA packaging formed during splicing acts as a regulator that shapes mRNA modifications.

2022-12-06 | GSE207663 | GEO

CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

Project description:The exon junction complex (EJC) is a central effector of mRNAs fate, linking nuclear processing to mRNA transport, translation and surveillance. Little is known about its transcriptome-wide targets. We used high-throughput sequencing after crosslinking and immunoprecipitation (HITS-CLIP) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks mainly located in spliced mRNAs. Most expressed exons harbour peaks equally distributed between the canonical EJC region ∼24 nucleotides upstream of exonic junctions and other non-canonical regions. Unexpectedly, both are preferentially associated to unstructured and purine-rich sequences containing the motif GAAGA, a potential binding site of EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation. To identify direct RNA binding sites of the EJC core component eIF4AIII, two biological CLIP-seq replicates were performed in HeLa cells. Additionally, mRNA-seq of the HeLa transcriptome was performed to normalize for the mRNA expression levels.

2012-10-21 | E-GEOD-40778 | biostudies-arrayexpress

Exclusion of m6A from splice-site-proximal regions by the exon-junction complex dictates m6A topologies and mRNA stability

Project description:N6-methyladenosine (m6A), a widespread destabilizing mark on mRNA, is non-uniformly distributed across the transcriptome, yet the basis for its selective deposition is unknown. Here, we uncover that m6A deposition is not selective. Instead, m6A distribution is exclusion-based: m6A-consensus harboring sites are methylated by default, unless they are within a window of up to ~200 nt from an exon-intron junction. A simple model which we extensively validate, relying exclusively on presence of m6A motifs and exon-intron architecture allows high accuracy recapitulation of experimentally-measured m6A profiles and of the vast majority of m6A hallmarks. Finally, we provide strong evidence that this exclusion is mediated by the exon-junction complex. Our findings establish a mechanism by which the memory of nuclear RNA splicing is covalently etched on an mRNA in the form of m6A, and determines its cytoplasmic stability, with broad implications on the regulation, function, and evolution of the exon-junction complex and m6A.

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Genome-wide identification of circRNAs translated by eIF4A3

Project description:Exon junction complexes (EJCs) deposited on spliced mRNAs play multifunctional roles in the regulation of gene expression. Whereas the formation and components of EJCs are well characterized, the underlying molecular mechanisms for gene regulation remain poorly understood. Here we find that a eukaryotic translation initiation factor (eIF) 4A3, a core component of EJC directly interacts with eIF3g, a subunit of eIF3 complex. This interaction serves as a linker between the EJC and eIF3 complex, consequently driving an internal ribosomal recruitment. Accordingly, artificially tethered EJC component or cellular EJC deposited on mRNA after splicing promotes internal initiation of translation in a way that is resistant to cellular stress induced by serum starvation. We also demonstrate that translatable endogenous or reporter circular RNAs depend on EJC for their association with polysomes. Our results uncover an internal initiation driven by EJC, expanding the protein-coding potential of human transcriptome including circular RNAs.

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Magoh, Y14, eIF4AIII interactomes by TurboID proximity labelling provide no evidence for a canonical EJC in (almost) intron-less trypanosomatids

Project description:In metazoans, mRNA quality is tightly monitored from transcription to translation. A key role lies with the exon junction complex (EJC) that is placed upstream of the exon-exon junction after splicing. The EJC inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and non-sense mediated decay (NMD, UPF1/2/3). The protozoan parasite Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of the three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested as an adaptation of the EJC function to mark trans-spliced mRNAs. Here we explore the interactome of Magoh, Y14, eIF4AIII in T. brucei by TurboID proximity labelling.

2024-03-25 | PXD050946 | Pride

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

Project description:To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here. Deep sequencing based profiling of EJC RNA footprints obtained by tandem RNA immunoprecipitation (RIPiT) of RNase I digested RNA:protein complexes.

2012-10-18 | E-GEOD-41154 | biostudies-arrayexpress

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

2012-10-18 | GSE41154 | GEO

PRPF8 iCLIP on HeLa cells in response to eIF4A3 Knockdown

Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.

2018-10-14 | E-MTAB-7269 | biostudies-arrayexpress

CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

2012-10-21 | GSE40778 | GEO

The exon junction complex is required for definition and excision of neighboring introns in Drosophila

Project description:Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity either from an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wildtype cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon de-silencing in EJC-depleted Drosophila ovaries. We propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. Our observations provide a powerful basis to dissect the molecular events that underlie the role of the EJC in splicing. Analysis of splicing defects in 3 knockdowns (siGFP [control], siACN, siTSU) in Drosohpila OSCs. PolyA RNA (biological duplicates) and total-RNA was sequenced on a Illumina HiSeq2000 in PE50 mode.

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