URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway

https://doi.org/10.1016/j.bbagrm.2020.194480Get rights and content

Highlights

  • UAP56 and URH49 form distinct Apo-form TREX and AREX complex, respectively.

  • ATP drives both complexes remodeling from Apo-form complex to ATP-TREX complex.

  • Selective mRNA export depends on Apo-form TREX and AREX complex.

  • Diversified mRNA export pathway is integrated into NXF1.

Abstract

The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.

Introduction

In eukaryotic cells, protein-coding genes are transcribed as a pre-mRNA in the nucleus and the pre-mRNA undergoes several RNA processing steps, such as 5′-capping, splicing and 3′-end processing. These gene expression processes are tightly coordinated with each other through mRNA-protein complex (mRNP) remodeling to achieve efficient and accurate gene expression [[1], [2], [3]]. After the mRNA processing steps, the mRNA-protein complex becomes mature and get ready to be exported from the nucleus to the cytoplasm.

The TREX complex, a evolutionarily conserved protein complex, is responsible for the integration of the several mRNA processing steps to export mRNA [4,5] [[6], [7], [8], [9]]. The human TREX complex consists of multiple subunits, including the RNA helicase UAP56/DDX39B, ALYREF/THOC4, CIP29/SARNP, CHTOP, PDIP3/POLDIP3, ZC11A/ZC3H11A and the THO subcomplex, which comprises THOC1/HPR1, THOC2 /hTHO2, THOC3/hTEX1, THOC5/fSAP79, THOC6/fSAP35 and THOC7/fSAP24 [[10], [11], [12], [13], [14]]. And the TREX complex is recruited to mRNA in a splicing-dependent manner, probably via the interaction between UAP56 and U2AF65, a spliceosome component required for 3′ splice site recognition [6,10,15]. The assembly of the TREX complex is dynamically associated with the ATPase cycle of UAP56. That is, CIP29, ALYREF, CHTOP, PDIP3 and ZC11A within the TREX complex are dependent on ATP binding onto UAP56 [11,12,16]. The ATPase and RNA helicase activity of UAP56 is promoted by CIP29, ALYREF and CHTOP [11,16,17]. UAP56 also functions to recruit both ALYREF and CHTOP onto mRNA [13]. The interaction of ALYREF, THOC5 and CHTOP with NXF1, which is an mRNA exporter at the final stage of the mRNA export process, triggers NXF1 association onto export competent mRNA [8,13,18]. Because the binding of ALYREF to UAP56 and NXF1 is mutually exclusive, UAP56 is removed from the TREX complex during NXF1 recruitment. Thus, the TREX complex acts as a binding platform for NXF1. In addition, the TREX complex is recruited to the 5′ cap site of the mRNA via an interaction between ALYREF and the cap-binding complex (CBP80 and CBP20) during splicing. This ensures mRNA export to the cytoplasm in a 5′-to-3′ direction [[19], [20], [21], [22]].

In the TREX complex, UAP56 homologues (Sub2 in S. cerevisiae, UAP56 in C. elegans, and Hel25E in D. melanogaster) are essential for each species, indicating the critical role of this RNA helicase in mRNA export [[23], [24], [25]]. In mammals, another Sub2 homolog, URH49/DDX39A has been identified to be involved in mRNA export [26], which shares sequences identity of 90% and similarity of 96% with UAP56. However, their target mRNAs are different at the genome-wide level [27]. UAP56 is continuously expressed during the cell cycle. In contrast, the expression level of URH49 is more dynamic observed in cultured mammalian cells, low in the quiescent phase but is increased during the cell proliferation phase [26], which indicates that URH49-dependent mRNA export is linked to the cell proliferation.

In our previous study, UAP56 forms the TREX complex with the THO subcomplex and ALYREF, while URH49 forms the AREX complex with CIP29 [27]. The differences in the complex formation between UAP56 and URH49 have been thought to associate with the selective recognition of a specific set of mRNA species. In contrast, conflicting results are also reported. The TREX component THOC2 has been shown to be associates with URH49 in vivo [11]. And other reports have demonstrated that UAP56 and URH49 directly interact with ALYREF as well as CIP29 [28,29]. To date, the molecular mechanism on the complex formation of the TREX complex and the AREX complex remains unsolved.

In the present report, we characterize the formation of the TREX complex and the AREX complex in the absence or presence of ATP. We show that both the TREX complex and the AREX complex have two forms. In Apo-form, the two complexes are defined by a distinct set of components. In ATP bound form, they share highly similar composition, designated as ATP-TREX complex. The addition of ATP drove both complexes remodeling from Apo-form complex to ATP-form complex. Furthermore, we confirm that the Apo-AREX complex is involved in the selective mRNA export. And URH49- and CIP29-dependent mRNA export are integrated with NXF1 upon the transition of Apo-AREX complex to ATP-TREX complex, which consistent with previous observation in UAP56-dependent mRNA export pathway. These results indicated that, in humans, the mRNA export pathway was diversified into two pathways as the formation of different Apo-form complex, which is integrated into NXF1 through the ATP-induced complex remodeling.

Section snippets

Cell culture

HeLa, MCF7, 293 Flp-In T-REx, H1299, HCT116WT and HCT116 p53-/- cells were maintained in Dulbecco's Modified Eagle's Medium (Wako, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum at 37 °C. 293 Flp-In T-REx cells stably expressing FLAG-UAP56, FLAG-URH49 and FLAG-CIP29 were obtained by the transfection of pcDNA5 FLAG-UAP56, pcDNA5 FLAG-URH49 or pcDNA5 FLAG-CIP29 in combination with pOG44, respectively. MCF7 cells stably expressing H2B-GFP were generated by H2B-EGFP

ATP binding remodels compositions of the Apo-TREX complex and the Apo-AREX complex into the ATP-TREX complex

To investigate the complex formation in the absence or presence of ATP, FLAG-UAP56 and FLAG-URH49 were stably expressed in 293 Flp-In T-REx cells respectively. We performed immunoprecipitation to detect the interaction of the target proteins in the NE prepared from FLAG-UAP56 or FLAG-URH49 expressed cells, using FLAG antibody fixed beads. As previously reported, UAP56 interacted with THOC1 and THOC5 even in the ATP depleted condition, while associated with ALYREF and CIP29 upon ATP addition [11

Discussion

UAP56 and URH49, closely related RNA helicases, have roles in mRNA maturation in the nucleus and export to the cytoplasm by forming the TREX complex and the AREX complex in human, respectively [27]. It has also been reported that UAP56 was associated with CIP29 in the presence of ATP. URH49 was associated with THOC2 and ALYREF in the presence of ATP but this association was not investigated in the absence of ATP [11]. Thus, the underlying molecular mechanism remains largely unsolved.

CRediT authorship contribution statement

Ken-ichi Fujita:Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing - original draft.Tomohiro Yamazaki:Data curation, Formal analysis, Investigation.Kotaro Harada:Formal analysis, Investigation.Shigeto Seno:Formal analysis, Investigation.Hideo Matsuda:Supervision.Seiji Masuda:Conceptualization, Funding acquisition, Project administration, Writing - original draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Dr. Robin Reed for NXF1 rabbit polyclonal antibodies, and Dr. Tomohiro Matsumoto for the general gift of expression plasmids, H2B-GFP, CFP-H2B, and YFP-tubulin.

Funding sources

This work was supported in part by grants-in-aid from JSPS KAKENHI (Grant Numbers JP2629053 and JP19H02884 to S.M and JP13J08335 to K.F). This work was also supported in part by grants-in-aid from The Kyoto University Foundation to S. M and The Sasakawa Scientific Research Grant from Japan Science Society to K.F.

References (64)

  • W. Jiang et al.

    PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis

    Mol. Cell

    (1998)
  • S. Fukuda et al.

    Cloning and characterization of a proliferation-associated cytokine-inducible protein, CIP29

    Biochem. Biophys. Res. Commun.

    (2002)
  • K. Kikuta et al.

    Clinical proteomics identified ATP-dependent RNA helicase DDX39 as a novel biomarker to predict poor prognosis of patients with gastrointestinal stromal tumor

    J. Proteome

    (2012)
  • K.G. Calderón-González et al.

    Determination of the protein expression profiles of breast cancer cell lines by quantitative proteomics using iTRAQ labelling and tandem mass spectrometry

    J. Proteome

    (2015)
  • C. Dominguez-Brauer et al.

    Targeting mitosis in cancer: emerging strategies

    Mol. Cell

    (2015)
  • T. Maniatis et al.

    An extensive network of coupling among gene expression machines

    Nature

    (2002)
  • S. Komili et al.

    Coupling and coordination in gene expression processes: a systems biology view

    Nat. Rev. Genet.

    (2008)
  • S. Jimeno et al.

    The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability

    EMBO J.

    (2002)
  • K. Stäßer et al.

    TREX is a conserved complex coupling transcription with messenger RNA export

    Nature

    (2002)
  • S. Masuda et al.

    Recruitment of the human TREX complex to mRNA during splicing

    Genes Dev.

    (2005)
  • J. Katahira et al.

    Adaptor Aly and co-adaptor Thoc5 function in the Tap-p15-mediated nuclear export of HSP70 mRNA

    EMBO J.

    (2009)
  • C.G. Heath et al.

    The role of TREX in gene expression and disease

    Biochem. J.

    (2016)
  • B. Chi et al.

    Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA

    Nucleic Acids Res.

    (2013)
  • K. Dufu et al.

    ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex

    Genes Dev.

    (2010)
  • E.G. Folco et al.

    The proteins PDIP3 and ZC11A associate with the human TREX complex in an ATP-dependent manner and function in mRNA export

    PLoS One

    (2012)
  • C. Te Chang et al.

    Chtop is a component of the dynamic TREX mRNA export complex

    EMBO J.

    (2013)
  • S. Younis et al.

    Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth

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

    (2018)
  • J. Fleckner et al.

    U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction

    Genes Dev.

    (1997)
  • C.-T. Chang et al.

    Chtop is a component of the dynamic TREX mRNA export complex

    EMBO J.

    (2013)
  • I. Taniguchi et al.

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

    Mol. Cell. Biol.

    (2008)
  • N. Viphakone et al.

    TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export

    Nat. Commun.

    (2012)
  • A.M. Gromadzka et al.

    A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

    Nucleic Acids Res.

    (2016)
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    Present address: Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan.

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