Structural and functional analysis of RNA and TAP binding to SF2/ASF

Tintaru, Aura; Hautbergue, Guillaume M.; Hounslow, Andrea M.; Hung, Ming‐Lung; Lian, Lu‐Yun; Craven, C. Jeremy; Wilson, Stuart A.

doi:10.1038/sj.embor.7401031

Cited by 73 publications

(144 citation statements)

References 23 publications

(41 reference statements)

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“…An arginine-rich motif is essential for TAP interaction with 9G8, SRp20, and SF2/ASF (12,13). Therefore, we assessed whether arginines present within REF amino acids 16-36 were required for TAP binding.…”

Section: Resultsmentioning

confidence: 99%

“…In this assay, the activity of an export factor is monitored by tethering it to an inefficiently exported reporter mRNA via bacteriophage MS2 coat protein (MS2) and RNA operator sequences (13) (Fig. 2E).…”

Section: Resultsmentioning

confidence: 99%

“…The shuttling SR proteins 9G8, SRp20, and SF2/ASF directly bind TAP by short arginine-rich peptides (12,13) and can function as export factors (14). Even in yeast, other proteins can recruit Mex67p to the mRNP, including Yra2p (15) and Npl3p.…”

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confidence: 99%

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Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Hautbergue

Hung

Golovanov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

226

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REF/Yra1p interacts with TAP/Mex67p, and in yeast this interaction leads to displacement of Sub2p from Yra1p (7). TAP heterodimerizes with p15 and binds nucleoporins through central and C-terminal domains (8), directing the mRNP to the nuclear pore and promoting transport to the cytoplasm. On the cytoplasmic side of the nuclear pore, Dbp5p triggers displacement of Mex67p from mRNA. Yra1p binds mRNA early during its nuclear maturation but is no longer bound once it reaches the nuclear periphery (9). Consistent with this finding, analysis of Balbiani ring pre-mRNPs shows that UAP56 and REF accompany the mRNP to the nuclear periphery where UAP56 and then REF dissociate during translocation through the pore (10).Although Yra1p is essential for yeast mRNA export, depletion of REF in higher eukaryotes does not block bulk mRNA export (11), suggesting that other proteins can fulfill this role and that there may be functional redundancy between export adaptors. The shuttling SR proteins 9G8, SRp20, and SF2/ASF directly bind TAP by short arginine-rich peptides (12, 13) and can function as export factors (14). Even in yeast, other proteins can recruit Mex67p to the mRNP, including Yra2p (15) and Npl3p.The fact that TAP binds RNA weakly in vitro led to the idea that export factors such as REF, which bind RNA avidly, bridge the interaction between TAP and mRNA, leading to the term mRNA export adaptors (15). However, both TAP and Mex67p are readily UV-cross-linked to mRNA in vivo (16)(17)(18), suggesting a direct stable interaction at some point during export. Here, we show that mRNA is handed over from export adaptors to TAP and that at least in vitro, export adaptors have the ability to enhance the RNA-binding activity of TAP. ResultsThe RNA-and TAP-Binding Sites on REF Overlap. In Saccharomyces cerevisiae, Mex67p binding to Yra1p triggers displacement of Sub2p (7), so we established whether this is the case for the mammalian orthologs. We examined whether UAP56 coimmunoprecipitated (Co-IP) with REF2-I (REF) in the presence of increasing amounts of TAP. This analysis revealed that TAP triggered dissociation of UAP56 from REF (Fig. 1A, lanes 5 and 6).We analyzed organization of the resulting REF-TAP-RNA ternary complex by examining how REF binds RNA. NMR

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Hautbergue

Hung

Golovanov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

162

226

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“…Assays were carried out as described in Hargous et al (2006) and Tintaru et al (2007), with the exception that renilla luciferase rather than ß-galactosidase was used for the normalization of transfection efficiency. For each transfection, 700 ng of each of the plasmids encoding the MS2-protein, 50 ng of luc-RRE firefly construct, and 5 ng of pRL-TK, a thymidine kinase renilla luciferase control vector, were cotransfected in 24-well plates.…”

Section: Tethered Mrna Export Assaymentioning

confidence: 99%

“…This raises the possibility that multiple and partially redundant adaptor proteins may be responsible for the recruitment of NXF1. Indeed, spliceosomal proteins, including U2AF35 (Zolotukhin et al, 2002) and some members of the SR family of splicing factors, were shown to interact with NXF1 and act as adaptors for NXF1-dependent export of poly(A) mRNAs (Huang and Steitz, 2001;Huang et al, 2003;Lai and Tarn, 2004;Hargous et al, 2006;Tintaru et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export

Ruepp

Aringhieri

Vivarelli

et al. 2009

MBoC

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Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3 end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3 end processing complex, CF I m 68, stimulates mRNA export. CF I m 68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF I m 68 may act as a novel adaptor for NXF1/TAP, we show that CF I m 68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF I m 68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3 end processing factor CF I m 68 in mRNA export. INTRODUCTIONThe removal of introns by splicing as well as cleavage and polyadenylation at the 3Ј end of RNA polymerase II primary transcripts (pre-mRNAs) are usually required before they can be exported from the nucleus as mature mRNAs (Erkmann and Kutay, 2004). This observation has suggested that transport factors interact with the RNA during premRNA processing. Indeed, recent discoveries have lent support to this hypothesis. The splicing reaction deposits on the mRNA a specific subset of proteins called the exon junction complex (EJC, for review see Tange et al., 2004). REF, a component of the EJC, facilitates mRNA export by interacting with the mRNA export factor NXF1 (also called TAP, for review, see Reed and Hurt, 2002). NXF1 was originally identified as the export receptor for type D retroviral RNAs that associate with NXF1 through a sequence-specific interaction with the constitutive transport element (CTE). However, NXF1 recruitment on cellular mRNAs requires adaptor proteins such as Aly/REF (hereafter named REF). In yeast Mex67 (the homolog of NXF1) is recruited by Yra1 (homolog of REF), which is also essential for the export of poly(A) RNA in Saccharomyces cerevisiae. In contrast in metazoans, REF is dispensable for bulk mRNA export. This raises the possibility that multiple and partially redundant adaptor proteins may be responsible for the recruitment of NXF1. Indeed, spliceosomal proteins, including U2AF35 (Zolotukhin et al., 2002) and some members of the SR family of splicing factors, were shown to interact with NXF1 and act as adaptors for NXF1-dependent export of poly(A) mRNAs (Huang and Steitz, 2001;Huang et al., 2003;Lai and Tarn, 2004;Hargous et al., 2006;Tintaru et al., 2007).Several observations have linked 3Ј end cleavage and polyadenylation to mRNA export (for review see Zhao et al., 1999). For example, RNA polymerase II reporter transcripts lacking a polyadenylation signal are retained in the nucleus of yeast cells. Positioning a...

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RNA –Protein Interactions: A New Approach for Drugging RNA Biology

Soueid,

Garner

2024

RNA as a Drug Target

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Structural and functional analysis of RNA and TAP binding to SF2/ASF

Cited by 73 publications

References 23 publications

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export

RNA –Protein Interactions: A New Approach for Drugging RNA Biology

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Structural and functional analysis of RNA and TAP binding to SF2/ASF

Cited by 73 publications

References 23 publications

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Mammalian pre-mRNA 3′ End Processing Factor CF Im68 Functions in mRNA Export

RNA –Protein Interactions: A New Approach for Drugging RNA Biology

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Mammalian pre-mRNA 3′ End Processing Factor CF I_m68 Functions in mRNA Export