In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

Custódio, Noélia; Carvalho, Célia; Condado, Inês; Antoniou, Michael; Blencowe, Benjamin J.; Carmo‐Fonseca, Maria

doi:10.1261/rna.5258504

Cited by 81 publications

(74 citation statements)

References 93 publications

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“…Additionally, there is a need for new assays to probe the connections between promoter regulation and premRNA processing decisions. As RNA Pol II-RNA binding protein complexes may be reorganizing during mRNA synthesis, these data are consistent with models in which cotranscriptional assembly of RNPs is a dynamic process (Mortillaro et al 1996;Wetterberg et al 2001;Custodio et al 2004;Ujvari and Luse 2004;Yu et al 2004). …”

Section: Significance Of Genomic Localization Of Rna Binding Proteinssupporting

confidence: 84%

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3Ј end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3Ј ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3Ј processing factor, CSTF2, had biases toward 3Ј ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA-protein complexes.[Supplemental material is available online at www.genome.org and http://silver.med.harvard.edu/brodsky/.] RNA binding proteins regulate many stages of RNA metabolism to help determine gene expression programs (for reviews, see Keene and Tenenbaum 2002;Hieronymus and Silver 2004). To prepare mRNA for export to and translation in the cytoplasm, many events including splicing, cleavage of the 3Ј end, and editing occur cotranscriptionally (Kornblihtt et al. 2004;Aguilera 2005a). After transcription, the mRNA-protein complex may reorganize as it is exported to the cytoplasm and prepared for translation (Fairman et al. 2004). Thus, significant efforts have been made to determine how RNA binding proteins interact at genes and/or mRNAs at various stages of mRNA metabolism (Tenenbaum et al. 2000;Brown et al. 2001;Hieronymus and Silver 2003;Ule et al. 2003;Yu et al. 2004). These early genomic efforts suggested that, similar to DNA binding proteins, functional groupings of genes are being bound by RNA binding proteins perhaps as post-transcriptional operons (Keene and Tenenbaum 2002). However, it is not known at which stage of RNA processing these operons are established.Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al. 1996;Yuryev et al. 1996;Kim et al. 1997). The CTD is phosphorylated as transcription begins. Following...

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Section: Significance Of Genomic Localization Of Rna Binding Proteinssupporting

confidence: 84%

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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“…60 and 61). Following splicing, the EJC is reorganized, and proteins that regulate mRNA nuclear export are recruited (33). Importantly, it has been demonstrated that the yeast homologues of Aly and Tap, Yra1 and Mex67, each associate with distinct and overlapping subsets of messages during export from the nucleus (62).…”

Section: Discussionmentioning

confidence: 99%

“…The TNF-␣ gene contains three introns and intron splicing results in deposition of exon junction complex (EJC) proteins on the message (30 -32). Following splicing, proteins of the EJC recruit proteins that regulate mRNA nuclear export (33). We hypothesized that EJC recruitment was required for the ERK regulation of TNF-␣ mRNA nuclear export.…”

mentioning

confidence: 99%

Extracellular Signal-regulated Kinase Regulation of Tumor Necrosis Factor-α mRNA Nucleocytoplasmic Transport Requires TAP-NxT1 Binding and the AU-rich Element

Skinner

Deleault

Fecteau

et al. 2008

Journal of Biological Chemistry

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Tumor necrosis factor-␣ (TNF-␣) production is regulated by transcriptional and posttranscriptional mechanisms. Lipopolysaccharide activates the NFB pathway increasing TNF-␣ transcription. Lipopolysaccharide also activates the mitogenactivated protein kinase pathways, resulting in stabilization and enhanced translation of the TNF-␣ message. In addition, nuclear export of the TNF-␣ mRNA is a posttranscriptionally regulated process involving the Tpl2-ERK pathway and requiring the presence of the TNF-␣ AU-rich element (ARE). We demonstrate that nuclear export of the TNF-␣ message requires not only the TNF-␣ ARE but also the interaction of the proteins TAP and NxT1, both of which are involved in nucleocytoplasmic transport of mRNA. Through the use of dominant negative ERK1 and ERK2, we establish that control of TNF-␣ mRNA nuclear export operates specifically through ERK1. Finally, we examined the role of two established TNF-␣ ARE-binding proteins, HuR and tristetraprolin, that shuttle between the nucleus and cytoplasm. These data demonstrate that neither tristetraprolin nor HuR is required for TNF-␣ mRNA export. It is unclear at this time if ARE-binding protein(s) directly interact with the TAP-NxT1 complex, if each complex is independently targeted by ERK1, or if only one complex is targeted.

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“…Mass spectrometry and Western blotting of purified spliceosomes revealed that REF is a component of H complex (30), suggesting that REF can associate with mRNAs in a splicing-independent manner. In situ analysis of green fluorescent protein-tagged REF showed its accumulation at sites of transcription (31), suggesting that REF binds to mRNA co-transcriptionally.…”

mentioning

confidence: 99%

The Interaction between Cap-binding Complex and RNA Export Factor Is Required for Intronless mRNA Export

Nojima

Hirose

Kimurâ

et al. 2007

Journal of Biological Chemistry

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RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited onIn eukaryotic cells, pre-mRNAs are primarily transcribed by RNA polymerase II (RNAPII); this process is followed by complex RNA processing steps that include capping, splicing, and polyadenylation to produce mature mRNAs. Recent studies have shown that individual events occurring during eukaryotic gene expression are coupled together under more elaborate regulatory controls than previously imagined (1-5). Coupling stimulates the rate and specificity of enzymatic reactions by tethering mechanisms to each other and to their substrates. Following processing, mRNA is exported as a large mRNA-protein complex (mRNP) 2 through the nuclear pore to the cytoplasm for subsequent translation.The transport of mRNA from the nucleus to the cytoplasm is linked to pre-mRNA splicing, especially in metazoans (6). Exon junction complexes (EJCs), which are deposited on mRNAs at specific sites relative to the exon junction as a consequence of splicing, form the basis of this connection (7,8). The EJC consists of four core proteins, eIF4A3 (9 -11), Y14 (7), Magoh (12, 13), and MLN51 (14), plus other auxiliary proteins including REF (7), UAP56 (15), RNPS1 (7), SRm160 (7), Pinin (16, 17), Acinus L (18), SAP18 (18), and hUpf3 (8,19,20). The recruitment of REF during mRNA biogenesis is thought to be responsible for the increased export of spliced mRNA (21). A DEAD box RNA helicase, UAP56, is required for the recruitment of REF to mRNA (15,22). Subsequently, UAP56 is displaced from REF by the mRNA export factor TAP (23). TAP forms a heterodimer with p15 that then directly interacts with the nuclear pore to facilitate mRNP transport into the cytoplasm (24).Although the above model explains the apparent link between splicing and RNA export in metazoans, the question of how intronless mRNAs, which lack EJC deposition, are exported to the cytoplasm naturally arises. Some intronless transcripts (e.g. histone H2A) have been reported to contain specific sequences that recruit export factors independently of splicing (25). SRp20 and 9G8, which belong to members of the evolutionarily conserved SR (serine/arginine-rich) protein family, specifically bind to a sequence in intronless mRNA and greatly facilitate the export of mRNA by recruiting TAP (26). However, the intronless mRNAs coding Ftz, dihydrofolate reductase, and ␤-globin, which lack such cis-acting sequences, can be effectively exported regardless of whether splicing has occurred (27)(28)(29) To clarify the recruiting mechanism of RNPs on mRNAs, we primarily developed a coupled in vitro transcription-splicing system. This in vitro system led us to discover a novel mechanism through which REF can associate with mRNA in a manner that is independent of splicing, instead of via the cap structure

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In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei

Abstract: ABSTRACT

Cited by 81 publications

References 93 publications

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Extracellular Signal-regulated Kinase Regulation of Tumor Necrosis Factor-α mRNA Nucleocytoplasmic Transport Requires TAP-NxT1 Binding and the AU-rich Element

The Interaction between Cap-binding Complex and RNA Export Factor Is Required for Intronless mRNA Export

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