ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56 - PubMed (original) (raw)

ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56

Ichiro Taniguchi et al. Mol Cell Biol. 2008 Jan.

Abstract

Loading of export factors onto mRNAs is a key step in gene expression. In vertebrates, splicing plays a role in this process. Specific protein complexes, exon junction complex and transcription/export complex, are loaded onto mRNAs in a splicing-dependent manner, and adaptor proteins such as Aly/REF in the complexes in turn recruit mRNA exporter TAP-p15 onto the RNA. By contrast, how export factors are recruited onto intronless mRNAs is largely unknown. We previously showed that Aly/REF is preferentially associated with intronless mRNAs in the nucleus. Here we show that Aly/REF could preferentially bind intronless mRNAs in vitro and that this binding was stimulated by RNA helicase UAP56 in an ATP-dependent manner. Consistently, an ATP binding-deficient UAP56 mutant specifically inhibited mRNA export in Xenopus oocytes. Interestingly, ATP activated the RNA binding activity of UAP56 itself. ATP-bound UAP56 therefore bound to both RNA and Aly/REF, and as a result ATPase activity of UAP56 was cooperatively stimulated. These results are consistent with a model in which ATP-bound UAP56 chaperones Aly/REF onto RNA, ATP is then hydrolyzed, and UAP56 dissociates from RNA for the next round of Aly/REF recruitment. Our finding provides a mechanistic insight into how export factors are recruited onto mRNAs.

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Figures

FIG. 1.

FIG. 1.

Recruitment of Aly/REF onto intronless mRNAs. (A) A mixture of 32P-labeled in vitro-transcribed RNAs containing two intronless mRNAs (DHFR and Ftz mRNAs), CTE, U1ΔSm, and U6Δss snRNAs was injected into the nuclei of Xenopus oocytes. The nuclear fraction was prepared after 1 h, and immunoprecipitation (IP) was performed with anti-Aly/REF monoclonal antibody (anti-REF, 11G5; ImmuQuest) (17) or control antibody (anti-Myc, 9E10; Sigma). RNA precipitated with each antibody was recovered and analyzed by denaturing PAGE and autoradiography. The input lanes contained 10% of the input mixture. (B) The same 32P-labeled RNA mixture as for panel A, except that tRNAPhe was supplemented, was incubated in 7.5% HNE, 12 mM HEPES-KOH (pH 7.9), 60 mM KCl, 1.6 mM MgCl2, 0.1 mM EDTA, 6% glycerol at 30°C for 30 min in the absence (−) or presence (+) of 2 mM ATP. After the incubation, immunoprecipitation was performed and the coprecipitated RNA was analyzed as described for panel A.

FIG. 2.

FIG. 2.

Recruitment of Aly/REF onto RNA by UAP56. (A) The same 32P-labeled RNA mixture as in Fig. 1A, except that CTE was omitted but intronless β-globin mRNA and U5ΔSm RNA were supplemented, was incubated with (+) or without (−) recombinant Aly/REF (10 nM) and recombinant Flag-tagged UAP56 (10 nM), either wild type (+) or GET mutant (GET, K95E), in either the absence (−) or presence (+) of 2 mM ADP, ATP, or ATP-γS at 30°C for 15 min. After the incubation, immunoprecipitation was performed with anti-Aly/REF antibody (anti-REF, 11G5; ImmuQuest) or anti-Flag antibody (M2; Sigma) and the coprecipitated RNA was analyzed as described for Fig. 1. (B) The same experiment as in panel A, lanes 1, 2, 4, and 5, except that a higher concentration (50 nM) of Aly/REF was employed. (C) Recombinant Flag-UAP56 (1 μg) and 3,000 Ci/mmol [α-32P]ATP (1 μl; Amersham) were incubated in the absence or presence of increasing concentrations of unlabeled ATP, ADP, ATP-γS, AMP-PNP, and AMP-PCP as competitors and irradiated with UV light as described in Materials and Methods. The cross-linked protein was analyzed by SDS-PAGE and autoradiography. (D) A UV cross-linking experiment similar to that shown in panel C was performed with wild-type UAP56 (UAP-WT), UAP56 LAT mutant (UAP-LAT, S228L), or UAP56 GET mutant (UAP-GET, K95E), without nucleotide competitors, in the absence (−) or presence (+) of yeast RNA (1 mg/ml; Sigma).

FIG. 3.

FIG. 3.

Effect of UAP56 proteins on RNA export. (A) A 32P-labeled RNA mixture containing intronless Ftz mRNA (Ftz), U1ΔSm, U6Δss RNAs, and tRNAPhe (top) or 32P-labeled pre-Ftz RNA (bottom) was microinjected into the nuclei of Xenopus oocytes with or without three doses of either UAP56 wild-type (WT) or UAP56-GET mutant (GET) proteins. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions immediately (lanes 1 and 2) or 3 h (lanes 3 to 16) after injection and analyzed by 8% denaturing PAGE followed by autoradiography. (B) Quantitation of relative RNA export efficiency shown in panel A.

FIG. 4.

FIG. 4.

Interaction of UAP56 with RNA and Aly/REF. (A) The same 32P-labeled RNA mixture as in Fig. 2A but supplemented with tRNAPhe was incubated with (+) or without (−) recombinant Flag-UAP56 (100 nM) in either the absence (−) or presence (+) of 2 mM ADP, ATP, or ATP-γS at 30°C for 15 min. After the incubation, immunoprecipitation was performed with anti-Flag antibody and the coprecipitated RNA was analyzed as described for Fig. 1. (B) Recombinant Flag-UAP56 wild type (WT) or GET mutant (GET) was incubated with GST alone (GST) or GST-Aly/REF (GST-REF) in the presence of RNase A and in the absence or presence of 2 mM ATP, ADP, or ATP-γS, and GST pull-down was performed as described in Materials and Methods. Flag-UAP56 proteins were visualized by Western blotting with anti-Flag antibody (M2).

FIG. 5.

FIG. 5.

ATPase activity of UAP56. (A) ATPase activity of recombinant Flag-UAP56 was measured as described in Materials and Methods, with or without a 100-nt RNA derived from the β-globin mRNA sequence and/or recombinant Aly/REF. Pi, released phosphate; ori, origin of chromatography. (B) Quantitation of relative ATPase activities from four independent experiments as described for panel A. Averages and standard errors are shown. (C) 35S-labeled Aly/REF protein produced in the E. coli T7 S30 system (Promega) was incubated with GST alone (GST; 300 pmol), GST-UAP56 (GST-UAP; 300 pmol), or GST-TAP231 (GST-TAP; 10 pmol) (12, 15) in the presence of RNase A and in the absence (lanes 2, 3, and 6) or presence (lanes 4, 5, 7, and 8) of recombinant TAP231 (10 and 100 pmol; lanes 4 and 5, respectively) or UAP56 (300 and 3,000 pmol; lanes 7 and 8, respectively), and GST pull-down was performed as described for Fig. 4B. 35S-labeled Aly/REF protein was visualized by fluorography.

FIG. 6.

FIG. 6.

A model of export factor recruitment to intronless mRNAs. Note that for the sake of simplicity, this model shows Aly/REF as the only adaptor protein to recruit TAP-p15, although this is not the case. See the text for details.

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