Accepted Manuscript Characterization of the nuclear import pathway for BLM protein Zhiqiang Duan, Jiafu Zhao, Houqiang Xu, Haixu Xu, Xinqin Ji, Xiang Chen, Jianming Xiong PII:

S0003-9861(17)30444-7

DOI:

10.1016/j.abb.2017.09.019

Reference:

YABBI 7566

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 4 July 2017 Revised Date:

18 September 2017

Accepted Date: 29 September 2017

Please cite this article as: Z. Duan, J. Zhao, H. Xu, H. Xu, X. Ji, X. Chen, J. Xiong, Characterization of the nuclear import pathway for BLM protein, Archives of Biochemistry and Biophysics (2017), doi: 10.1016/j.abb.2017.09.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

BLM enters the nucleus via the importin β1, RanGDP and NTF2 dependent pathway. (i)

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Importin β1 forms a binary complex with BLM in the cytoplasm. (ii) The ternary complex is

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formed by binging of RanGDP to importin β1 and stocks at the nuclear pore complex (NPC). (iii) NTF2 binds RanGDP and triggers the nuclear import of the ternary complex. (iv) Inside the nucleus regulator of chromosome condensation 1 (RCC1) exchanges GDP with GTP on

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Ran, generating importin β1/RanGTP complex and releasing BLM and NTF2.

ACCEPTED MANUSCRIPT 1

Characterization of the nuclear import pathway for BLM protein

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Zhiqiang Duana,b, Jiafu Zhaoa,b, Houqiang Xua,b, Haixu Xuc, Xinqin Jib, Xiang Chena,b,

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Jianming Xiongb

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a

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Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China

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b

College of Animal Science, Guizhou University, Guiyang 550025, China

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c

College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China

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Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau

*Corresponding author: Houqiang Xu.

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Mailing address: College of Animal Science, Guizhou University, 14 Xiahui Road, Huaxi

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District, Guiyang, Guizhou Province, 550025, China.

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Tel.: +86 851 88298005

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Fax: +86 851 88298003

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E-mail: [email protected]

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Abbreviations: RQC, RecQ C-terminal; HRDC, helicase-and-ribonuclease D-C-terminal;

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NLS, nuclear localization signal; NPC, nuclear pore complex; HTLV-1, human T-cell

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leukemia virus type 1; SREBP2, sterol regulatory element-binding protein 2; HPV16, human

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papillomavirus Type 16.

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Abstract Numerous studies have shown that nuclear localization of BLM protein, a member of the

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RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA

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recombination, replication and transcription, but the mechanism by which BLM protein is

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imported into the nucleus remains unknown. In this study, the nuclear import pathway for

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BLM was investigated. We found that nuclear import of BLM was inhibited by two

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dominant-negative mutants of importin β1 and NTF2/E42K, which lacks the ability to bind

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Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in

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GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using

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importin β1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had

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direct binding to importin β1 through its NLS domain with the 14-16 HEAT repeats of

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importin β1. Furthermore, importin β1, Ran or NTF2 depletion by siRNA disrupted the

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accumulation of BLM protein in the nucleus. These results showed that BLM enters the

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nucleus via the importin β1, RanGDP and NTF2 dependent pathway, demonstrating for the

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first time the nuclear trafficking mechanism of a DNA helicase.

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1. Introduction RecQ helicases are a family of DNA unwinding proteins evolutionarily conserved from

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bacteria to mammals that play essential roles in different DNA metabolic processes [1, 2].

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Unlike the genomes of bacteria and yeast typically encode only one RecQ homolog, the

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human genome encodes five different RecQ homologs including RECQ1, BLM, WRN,

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RECQ4 and RECQ5 [3]. Many studies have reported the existence of a highly conserved

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ATPase domain that acts as an ATP-dependent DNA translocation module, and a RecQ

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C-terminal (RQC) domain that regulates the binding of helicases to G quadruplex (G4) DNA

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and stabilizes binding to other DNA structures, in the five human RecQ helicases [4-6]. In

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addition, they all have common nuclear localization features mediated by their own nuclear

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localization signal (NLS) [7-11]. However, there is a unique helicase-and-ribonuclease

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D-C-terminal (HRDC) domain that plays an important role in promoting the localization to

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specific DNA lesions found in BLM and WRN [12, 13].

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In recent years, numerous studies have demonstrated that nuclear import of proteins

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mediated by NLS is essential for the regulation of protein function in various biological

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processes, such as cell-cycle progression, signal transduction, or the replication cycle of

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diverse viruses [14-16]. Nucleocytoplasmic trafficking of proteins across the nuclear

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membrane occurs through nuclear pore complex (NPC) [17]. However, nuclear import of

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proteins through the NPC can occur either through passive diffusion, or by an active process

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facilitated by the NLS recognized by the transport receptor proteins importin α and/or

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importin β [18, 19]. The basic paradigm for nuclear import is that importin α binds to the

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cargo’s NLS and importin β directs the docking of the importin α-cargo complex to the

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ACCEPTED MANUSCRIPT cytoplasmic side of the NPC. The translocation through the pore of RanGDP conjunct ternary

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complex is mediated by NTF2 (p10) via interactions with nucleoporins at the NPC. Once

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inside the nucleus, binding of RanGTP to importin β causes the dissociation of the ternary

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complex [20, 21]. Interestingly, more and more studies have focused on alternative import

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mechanisms that involve importin β-related proteins in the absence of importin α [19, 22]. For

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example, importin β1 can function without adapters in import of different cellular or viral

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proteins containing Arg/Lys-rich NLS [23, 24], and importin β2 directly mediates the nuclear

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import of some proteins via interaction with their Gly/Asn-rich NLS [25].

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Among the five human RecQ helicases, BLM protein has a molecular mass of

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approximately 159 kDa, and is demonstrated to enter the nucleus through a classical

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Arg/Lys-rich NLS (1344RSKRRK1349) [8]. Many studies have been concerning about the

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functions of BLM in the nucleus and nucleolus [26-28], but there is little information

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available regarding the mechanism by which BLM protein is imported into the nucleus and

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nucleolus. In the present study, the nuclear import pathway for human BLM protein was

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investigated. We demonstrated that importin β1, RanGDP and NTF2 were sufficient to

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mediate the nuclear import of BLM protein. Moreover, the BLM protein had direct binding to

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importin β1 through its NLS region interacting with the 14-16 HEAT repeats of importin β1.

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These results demonstrate for the first time the important role of importin β1, RanGDP and

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NTF2 in transporting BLM protein into the nucleus and may have implications for elucidating

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the nuclear import mechanism of the other human RecQ helicases.

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2. Materials and methods

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2.1. Cells and antibodies

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ACCEPTED MANUSCRIPT PC-3, HeLa and HEK-293T cells were purchased from Stem Cell Bank, Chinese

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Academy of Sciences. PC-3 cells were maintained in DMEM/F12 (1:1) medium, and HeLa

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and HEK-293T cells were maintained in DMEM medium, supplemented with 10% fetal

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bovine serum and 1% penicillin and streptomycin at 37℃ in a humidified incubator with 5%

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CO2. Primary antibodies mouse anti-GFP monoclonal antibody (sc-9996), mouse anti-GST

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monoclonal antibody (sc-374171), mouse anti-Myc monoclonal antibody (sc-40), mouse

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anti-actin monoclonal antibody (sc-8432) were purchased from Santa Cruz Biotechnology

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Company (USA). Mouse anti-importin β1 monoclonal antibody (ab2811), rabbit anti-Ran

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polyclonal antibody (ab53775), and rabbit anti-NTF2 antibody (ab137192) were purchased

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from Abcam (USA).

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2.2. Plasmid constructions

All enzymes used for cloning procedures were purchased from Thermo Fisher. The coding

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region of BLM1-1417 and BLM642-1417 was amplified from the cDNA derived from PC-3 cells

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and

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pEGFP-BLM642-1417, respectively. Plasmid pGEX-6p-BLM642-1417 and pGEX-BLM642-1417-GFP

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was constructed by subcloning BLM642-1417 gene into pGEX-6p-1 and pGEX-GFP,

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respectively. The importin β1 ORF was amplified and subcloned into pCMV-Myc and

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pET-32a(+) to yield pCMV-Myc-importin β1 and pET-32a-importin β1, respectively.

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Annealed oligonucleotides encoding the M9M or Bimax2 were inserted into pDsRed-C1

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(Clontech) to generate plasmid pDsRed-M9M and pDsRed-Bimax2, respectively. Dominant

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negative (DN) mutant RanGTP (Ran/Q69L), DN mutant NTF2 (NTF2/E42K), DN importin

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α5, DN importin β1 were subcloned into pDsRed-C1 to generate pDsRed-Ran/Q69L,

into

pEGFP-C1

(Clontech)

to

create

pEGFP-BLM1-1417

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ACCEPTED MANUSCRIPT pDsRed-NTF2/D23A, pDsRed-DN-importin α5 and pDsRed-DN-importin β1, respectively.

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All the recombinant plasmids were confirmed by PCR, restriction digestion and DNA

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sequencing. Primers used in this study are available upon request.

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2.3. Plasmid transfection and fluorescence microscopy

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For the transfection experiments, 3×105 HEK-293T cells were grown to 80% confluence

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in 35-mm-diameter dishes and then co-transfected with a total of 3 µg DNA using the

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FuGENE

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recommendations.

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phosphate-bufferd saline (PBS), fixed with 4% paraformaldehyde for 20 min, permeabilized

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with 0.25% Triton X-100 in PBS for 5 min, and then counterstained with DAPI (Sigma) to

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detect nuclei. Fluorescent images were obtained under a Nikon fluorescence microscope

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(Japan). Image analysis and merging of images were done with Adobe Photoshop 7.0

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software’s (Adobe Systems, CA, USA).

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2.4. Bacterial expression and protein purification

Thirty-six

Reagent hours

(Roche) after

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to

the

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manufacturer’s rinsed

with

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His-tagged importin β1 was expressed in E. coli BL21 (DE3) (4 h induction with 0.5 mM

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IPTG at 28℃), and the soluble His-tagged protein was purified on Ni-NTA His Bind Resin.

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GST-BLM642-1417-GFP, GST-BLM642-1417 fusion proteins were expressed in E. coli BL21 (DE3)

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(4 h induction with 1 mM IPTG at 30℃), and the soluble GST-fusion proteins were purified

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on Glutathione-Sepharose beads. GST-NTF2 was expressed in E. coli BL21 (DE3) (3 h

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induction with 1 mM IPTG at 37 ℃ ), and the soluble protein was purified on

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Glutathione-Sepharose beads. NTF2 was obtained by cleaving the GST-NTF2 fusion protein

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with biotinylated thrombin, followed by removal of the GST by binding to

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ACCEPTED MANUSCRIPT Glutathione-Sepharose beads and the thrombin by binding to Streptavidin-containing beads.

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Recombinant human Ran and Ran/Q69L proteins were expressed, purified, and charged with

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GDP or GTP, respectively, as previously described [29]. The purity and proteolytic

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degradation of the obtained proteins were checked by SDS-PAGE and Coomassie blue

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staining. The purified proteins were dialyzed in transport buffer containing protease inhibitors

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and then stored in aliquots at -80℃ until use.

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2.5. In vitro nuclear import assays

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In vitro nuclear import assays in digitonin-permeabilized cells were performed as

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previously

[30].

Briefly,

subconfluent

HeLa

cells

were

grown

on

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poly-L-lysine-coated glass coverslips for 1 day and then permeabilized with 70 µg of

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digitonin/ml for 5 min on ice. The digitonin-permeabilized HeLa cells were rinsed twice with

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transport buffer and then incubated for 15 min at room temperature with the import mixture.

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Import reactions contained an energy regenerating system (0.5 mM GTP, 5 mM

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phosphocreatine, and 0.4 U of creatine phosphokinase), plus various transport factors (0.5 µM

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importin β1; 1 µM NTF2; 3 µM RanGTP; 3 µM RanGDP; 3 µM RanQ69LGTP), plus the

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GST-BLM642-1417-GFP fusion protein (0.5 µM). The final reaction volume was adjusted to 20

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µL with transport buffer. After incubation, the cells were washed with transport buffer and

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fixed with 3.7% formaldehyde on ice followed by methanol for 5 min at -20℃. After three

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washes with transport buffer, the nuclei were identified by DAPI staining. Nuclear import was

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analyzed with a Nikon fluorescence microscope. Quantitation of nuclear import was done by

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measuring the fluorescence of 25 nuclei with IPLAB software.

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2.6. In-solution binding assays

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ACCEPTED MANUSCRIPT The interactions between GST-BLM642-1417 or GST and His-importin β1, RanGDP, NTF2

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were investigated using in-solution binding assays as previously described [29]. Briefly,

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GST-BLM642-1417 or GST immobilized on Glutathione-Sepharose beads (3 µg protein/10 µl

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beads), were incubated under rotation for 30 min at room temperature with His-importin β1 (3

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µg), and/or RanGDP (3 µg) plus NTF2 (3 µg) in transport buffer (total volume 40 µl). After

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incubation, the beads were washed twice with transport buffer and the bound proteins were

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eluted with 1×SDS-PAGE sample loading buffer and analyzed by SDS-PAGE followed by

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Coomassie blue staining.

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2.7. Protein interaction assays

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For co-immunoprecipitation assays, HEK-293T cells grown in 35-mm-diameter dishes

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were transfected with plasmid pEGFP-BLM642-1417. At 36 h post-transfection, cells were

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washed twice with PBS and lysed with immunoprecipitation buffer (Invitrogen). After

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centrifugation, the supernatant was incubated with anti-importin β1, or anti-GFP antibody for

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2 h. The immune complexes were recovered by adsorption to protein A+G Sepharose (Sigma)

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overnight at 4℃. After five washes in immunoprecipitation buffer, the immunoprecipitates

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were analyzed by Western blotting using anti-GFP or anti-importin β1 antibody.

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For GST pull-down assays, GST-BLM642-1417 or GST (a negative control) protein was

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immobilized on Gluthatione-Sepharose beads (1 µg/µl). After washing with transport buffer,

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the protein immobilized beads were incubated with purified His-importin β1 (3 µg) or

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exogenous Myc-importin β1 truncations derived from plasmids-transfected HEK-293T cells

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for 2 h at 4℃. The beads were then washed three times with transport buffer and the bound

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proteins were eluted from the beads, size-fractionated by SDS-PAGE, and immunoblotted for

ACCEPTED MANUSCRIPT importin β1 using anti-importin β1 antibody. In turn, His pull-down assay using His*Bind

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Resin-binded His-importin β1 protein and purified GST-BLM642-1417 or exogenous

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EGFP-BLM642-1417 protein derived from pEGFP-BLM642-1417 transfected HEK-293T cells was

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carried out as described above.

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2.8. siRNA treatment and fluorescence microscopy

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Human importin β1 siRNA (sc-35736), Ran siRNA (sc-36382), NTF2 siRNA (SC-36105)

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and Control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology Company.

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For examining the effect of siRNA-mediated knockdown of importin β1, Ran or NTF2

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protein on the nuclear localization of EGFP-BLM642-1417 fusion protein, low-passage

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HEK-293T cells grown on 35-mm-diameter dishes at a confluence of 80% were

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co-transfected with the siRNA and pEGFP-BLM642-1417. After 48 h transfection, the

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knockdown efficiency and the localization of EGFP-BLM642-1417 were checked by Western

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blot analysis and fluorescence microscopy, respectively. The transfected cells were

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counterstained with DAPI to detect nuclei. The subcellular localization patterns were

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quantified by counting 100-200 cells, and the predominant pattern was described and reported

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as a percentage of the total number of cells counted.

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3. Results

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3.1. EGFP-tagged BLM1-1417 and BLM642-1417 localize in the nucleus

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Previous studies have demonstrated that GFP-tagged BLM has the same enzymatic

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activity as normal BLM [31], and the amino acid residues 642-1290 that contain RQC and

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HRDC domains is the core region of BLM [12, 32]. Here, we investigated the subcellular

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localization of BLM1-1417 and BLM642-1417 containing the core region and NLS. Plasmids

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cells and expression of these proteins was first verified by Western blot analysis (Fig. 1A).

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The subcellular localization of EGFP and EGFP-tagged proteins was examined by

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fluorescence microscopy. As shown in Fig. 1B, EGFP alone was distributed in both the

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nucleus and cytoplasm, whereas the EGFP-tagged BLM1-1417 and BLM642-1417 were

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predominantly localized in the nucleus with concentrated foci, which is consistent with the

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previous findings [8, 33]. These results suggested that the fragment of BLM642-1417 had the

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same nuclear localization pattern and could be used to replace the full-length BLM1-1417 to

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study the nuclear localization mechanism of BLM.

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3.2. Nuclear import of BLM is inhibited by importin β1 and NTF2 mutants

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To identify the cellular transporter responsible for BLM nuclear targeting and further

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characterize the nuclear import pathway of BLM, two dominant-negative (DN) mutants of

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importin α5 (DN-importin α5) [34] and importin β1 (DN-importin β1) [35], which lack the

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ability to bind importin β and Ran, respectively, and nuclear import inhibitors M9M [36] or

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Bimax2 [37] that are specific for the transportin-1 pathway or the importin α1, α3, α6 and α7

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pathways, respectively, were introduced to determine whether they are required for the

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nuclear import of BLM protein. The results showed that HEK-293T cells were co-transfected

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with plasmid pEGFP-BLM642-1417 and plasmids encoding DsRed-DN-importin α5 or

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DsRed-M9M or DsRed-Bimax2 did not impair the nuclear transport of BLM642-1417, while

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co-expression of DsRed-DN-importin β1 significantly blocked the nuclear import of

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BLM642-1417 (Fig. 2A). In addition, RanGTP mutant (Ran/Q69L) [38], which is deficient in

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GTP hydrolysis, and NTF2 mutant (NTF2/E42K) [39, 40], which fails to transport RanGDP

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ACCEPTED MANUSCRIPT into nucleus, were also introduced to determine their role in the nuclear transport of BLM

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protein. As shown in Fig. 2B, co-expression of DsRed-Ran/Q69L with EGFP-BLM642-1417 did

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not change the nuclear localization of EGFP-BLM642-1417, whereas co-expression of

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DsRed-NTF2/E42K caused the localization of EGFP-BLM642-1417 to the cytoplasm. These

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results indicated that nuclear import of BLM might require the importin β1, NTF2 and was

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independent of GTP hydrolysis by Ran.

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3.3. Nuclear import of BLM requires importin β1, RanGDP and NTF2

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Next, in vitro nuclear import assays were performed to confirm whether importin β1,

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RanGDP and NTF2 are sufficient for BLM nuclear import. Results showed that the

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GST-BLM642-1417-GFP fusion protein was efficiently imported into the nucleus of

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digitonin-permeabilized cells in the presence of exogenous cytosol. Importin β1 alone was

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capable of only limited nuclear import of BLM, but importin β1 in the presence of RanGTP

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inhibited

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GST-BLM642-1417-GFP/importin β1 complex. In addition, RanGDP in the presence of its

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nuclear carrier NTF2 showed no nuclear translocation, while combination of importin β1 and

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RanGDP plus NTF2 exhibited the same level of nuclear accumulation as that seen when

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cytosol was added. Importantly, replacement of RanGDP with RanQ69LGTP led to

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perinuclear accumulation of BLM (Fig. 3A).

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suggesting

that

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dissociate

the

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Because addition of exogenous RanGDP and NTF2 was sufficient for nuclear import of

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BLM in digitonin-permeabilized cells, we investigated if BLM directly binds to the

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RanGDP/NTF2 complex. The results of in-solution binding assays showed that when

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GST-BLM642-1417 immobilized on Glutathione-Sepharose beads was incubated with

ACCEPTED MANUSCRIPT His-importin β1 or RanGDP/NTF2 complex, GST-BLM642-1417 binded to His-importin β1 but

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failed to bind RanGDP/NTF2 complex (Fig. 3B, lanes 1 and 2). However, when immobilized

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GST-BLM642-1417 was incubated with His-importin β1 and RanGDP/NTF2 complex, both

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His-importin β1 and RanGDP/NTF2 complex were detected (Fig. 3B, lane 3). As control,

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His-importin β1 or/and RanGDP/NTF2 complex did not bind to GST (Fig. 3B, lanes 4 to 6).

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These results clearly demonstrated that importin β1, in association with RanGDP/NTF2

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complex, were the components required to mediate the active nuclear import of BLM.

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3.4. BLM interacts with importin β1 in vivo and in vitro

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Numerous studies have demonstrated that importin β1 can directly recognize and bind

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different types of cargo NLSs and mediate their nuclear import [19, 41]. To determine

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whether BLM interacts with importin β1 in vivo and in vitro, we first performed

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co-immunoprecipitation assay with HEK-293T cells transiently transfected with plasmids

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expressing EGFP or EGFP-tagged BLM642-1417. As shown in Fig. 4A, EGFP-BLM642-1417 but

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not EGFP was detected in anti-importin β1 immunoprecipitates. In turn, importin β1 was also

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detected in the anti-GFP immunoprecipitates from the transfected cells overexpressing

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EGFP-BLM642-1417 (Fig. 4B), suggesting that BLM is associated with importin β1 in vivo. To

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further verify the physical interaction between BLM and importin β1, we examined their

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binding activities utilizing GST and His pull-down assays. A protein binding assay of

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GST-BLM642-1417 to His-importin β1 showed that His-importin β1 protein was pulled-down by

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GST-BLM642-1417 protein but not by GST (Fig. 4C). In the reciprocal experiment,

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GST-BLM642-1417 protein could also bind to His-importin β1 protein, but not His (Fig. 4D),

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indicating that BLM physically interacts with importin β1 in vitro.

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3.5. Mapping interaction domains between BLM and importin β1 To determine the domains involved in BLM and importin β1 interaction, a series of BLM

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and importin β1 deletion mutants were constructed to test their binding activities utilizing

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pull-down assays (Fig. 5A and B, upper panels). The expression of EGFP-BLM642-1417 and

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EGFP-BLM642-1417/NLSm in plasmid-transfected HEK-293T cells was detected by Western

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blot analysis (Fig. 5A, Input). Binding studies showed that the NLS region of BLM642-1417 was

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essential for interaction with importin β1, since EGFP-BLM642-1417/NLSm lost its binding

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ability to importin β1 (Fig. 5A, lower panel).

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Various functional domains have been identified within importin β1 protein [42, 43],

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including the IBN_N domain, RanGTP binding domain, and 19 HEAT repeats. To map the

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domain of importin β1 critical for BLM interaction, similar GST pull-down approach was

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employed. The expression of Myc-importin β1 and its deletion mutants was also detected by

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Western blotting (Fig. 5B, Input). Binding results showed that only the mutants of

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Myc-importin β1 containing the residues 600-724 could be pulled-down by GST-BLM642-1417

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(Fig. 5B, lower panel), demonstrating that the 14-16 HEAT repeats of importin β1 was

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essential for its interaction with BLM.

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3.6. Importin β1, Ran or NTF2 depletion disrupts the nuclear import of BLM

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The physical interaction between BLM and importin β1, and between importin β1 and

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Ran/NTF2 complex suggest that importin β1, Ran and NTF2 might regulate the nuclear

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localization of BLM. To further provide the evidence, we tested whether importin β1, Ran or

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NTF2 depletion disrupts the nuclear localization of BLM. To this end, the specifically

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down-regulated of importin β1, Ran or NTF2 in HEK-293T cells was verified using RNA

ACCEPTED MANUSCRIPT interference, and then the localization of EGFP-BLM642-1417 was analyzed under a

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fluorescence microscope. Western blotting analysis confirmed that the expression of importin

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β1, Ran or NTF2 was significantly reduced after transfection with importin β1, Ran or NTF2

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siRNA, respectively (Fig. 6A). In parallel, co-expression of the indicated siRNA and

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pEGFP-BLM642-1417 resulted in the decreased accumulation of EGFP-BLM642-1417 in the

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nucleus, whereas nuclear localization of EGFP-BLM642-1417 was not affected when control

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siRNA was co-tranfected (Fig. 6B and C). Together, these results demonstrated that importin

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β1, Ran and NTF2 were all required for BLM to accumulate in the nucleus.

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4. Discussion

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Up to now, the nuclear import mechanism of only two RNA helicases is clarified [44, 45].

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Many studies point to a nuclear role of BLM in DNA metabolism and stability [3, 46], and the

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nuclear localization of BLM is dependent on NLS [8], but so far the nuclear localization

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mechanism of BLM still remains unknown. Previous studies have demonstrated that nuclear

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import of most cargo proteins requires both importin α and importin β, with importin α as the

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adaptor between importin β and the cargo proteins [18, 47]. However, an increasing number

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of studies have shown that several proteins can undergo nuclear import via direct binding to

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importin β without the participation of importin α. These include the Rex protein of human

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T-cell leukemia virus type 1(HTLV-1) [23], HIV-1 Tat and Rev proteins [48], cyclin B1-Cdc2

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[49], Smad3 [50], sterol regulatory element-binding protein 2 (SREBP2) [51], human

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papillomavirus Type 16 (HPV16) E6 protein [52], Snail [53], human sexual regulator DMRT1

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[54], TopBP1 [55]. Among these, the nuclear import of HTLV-1 Rex, HIV Tat and Rev,

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Smad3, SREBP2 and Snail also requires RanGTP, but the nuclear import of HPV16 E6

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ACCEPTED MANUSCRIPT requires RanGDP and NTF2. Interestingly, Takizawa et al. [49] found that the nuclear import

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of cyclin B1-Cdc2 does not require Ran. These results reveal the diverse nuclear transport

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pathways mediated by importin β1. In the present study, we showed that the nuclear import of

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human BLM protein required importin β1, RanGDP and NTF2, and did not need to bind

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RanGTP to trigger the dissociation of importin β1 and BLM, demonstrating for the first time

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the nuclear trafficking mechanism of a DNA helicase.

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It is reported that Arg/Lys-rich NLSs within cargo proteins are served as binding sites for

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the recognition and binding of importin α or importin β [18, 19]. Generally, classical NLSs

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including monopartite and bipartite NLSs are imported by importin α/β heterodimer, while

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non-classical NLSs can be more complex in sequence, length and amino acid composition

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that are imported by importin β. However, more and more studies have proven that classical

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NLSs can also be recognized and binded by importin β1, such as the NLS of HTLV-1 Rex

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(RRRPRRSQRKR) [23], HIV-1 Tat (RKKRRQRRR) and Rev (RQARRNRRRR) [48],

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Smad3 (KKLKK) [50], and TopBP1 (RKRK) [55]. Our results similarly found that the

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classical NLS (RSKRRK) of BLM was critical for its interaction with importin β1 without

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preferentially recognized by importin α. It is known that various types of importin α are

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expressed at widely divergent levels in different tissues and also display very different

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affinities for distinct NLSs [56, 57]. In recent years, some studies even demonstrated that

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importin α protein can act as negative regulators for nuclear import of cargo proteins [58, 59].

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Therefore, potential difficulties in achieving efficient nuclear import of BLM in all of the

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various tissues could be avoided by binding importin β1 directly, rather than relying on one or

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more forms of importin α as an intermediary.

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ACCEPTED MANUSCRIPT As adaptor of cargo proteins, importin β1 contains importin-β N-terminal (IBN_N)

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domain at the N-terminus and several “HEAT repeat” motifs that mostly occupy the

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C-terminal portion [60]. The HEAT repeat motifs are able to form different conformations in

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different functional states, which facilitate the accommodation of their binding partners by an

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induced fit type of mechanism [21, 43, 61]. Numerous studies have shown that the HEAT

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repeats of importin β1 can provide rich binding regions for its interaction with cargo proteins,

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such as importin β1 binds PTHrP [62], Snail [63], SREBP2 [64] and TopBP1 [55] using

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B-helices spanning HEAT repeats 2-11, 5-14, 7-17 and 18-19, respectively. Although the

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HEAT repeats used to bind the three cargo proteins (2-11 for PTHrP, 5-14 for Snail, and 7-17

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for SREBP2) overlap, the binding mechanism in each is distinctly different [61]. Meanwhile,

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the binding regions of cargo proteins overlap with the region binding RanGTP (HEAT repeats

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8-10), indicating that cargo proteins-importin β1 heterodimer requires a large contact area and

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the ability for the complex to be disassembled by RanGTP binding upon entry to the nucleus.

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This is in agreement with the previous experimental results [62-64]. Unlike the above proteins,

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we found that the HEAT repeats 14-16 of importin β1 was responsible for interaction with

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BLM, and the nuclear import of BLM was dependent on RanGDP and NTF2, further

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confirming that cargo proteins that interacted with the RanGTP binding region of importin β1

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required RanGTP for nuclear targeting.

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In addition to the nuclear localization of BLM via classical NLS, one recent study has

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found that two serines within BLM (S1342 and S1345) are critical for its nucleolar

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localization [65], but the nucleolar localization signal of BLM is different from the classical

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nucleolar localization signal composed of basic amino acids [66]. Even so, the mechanisms

ACCEPTED MANUSCRIPT regulating localization of BLM to the nucleus and nucleolus still remain unknown. Given the

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importance of nuclear and nucleolar localization of BLM, we first investigated the nuclear

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localization mechanism of BLM and demonstrated that BLM entered the nucleus via the

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importin β1, RanGDP and NTF2 dependent pathway, which will provide useful implications

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for elucidating the nuclear import mechanism of other species’ BLM protein as well as other

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human RecQ helicases.

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Disclosures

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The authors have no conflict of interest to disclose.

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Acknowledgements

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This work was supported by the National Natural Science Foundation of China (31360215),

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the Talents Fund from Governor of Guizhou Province (QSZHZ-2012-60), the Science and

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Technology Fund of Guizhou Province (QKH-2015-2054) and the Scientific Research Project

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of Guizhou University Talents Fund (GDRJHZ-2014-10).

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ACCEPTED MANUSCRIPT Fig.1. Subcellular localization of EGFP-tagged BLM1-1417 and BLM642-1417. HEK-293T cells were transiently transfected with the plasmids expressing EGFP-tagged BLM1-1417 and BLM642-1417. After 36 h transfection, expression of the fusion proteins was

was used to stain the nuclei. Original magnification was 1×200.

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verified by Western blot analysis (A) and fluorescence microscopy (B), respectively. DAPI

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Fig.2. Nuclear import of BLM protein is inhibited by importin β1 and NTF2/E42K mutants.

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HEK-293T cells were transiently co-transfected with the plasmid pEGFP-BLM642-1417 (A), pEGFP-NLSBLM (B) or pEGFP-OGFr (C) and plasmids encoding DsRed-DN-importin α5, DsRed-DN-importin

β1,

DsRed-M9M,

DsRed-Bimax2,

DsRed-Ran/Q69L

or

DsRed-NTF2/E42K, respectively. After 36 h transfection, the subcellular localization of

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EGFP-BLM642-1417, EGFP-NLSBLM and EGFP-OGFr was observed under fluorescence microscope. DAPI was used to stain the nuclei. Original magnification was 1×200. In parallel, the nuclear and cytoplasmic fractions derived from plasmid-transfected HEK-293T cells were

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extracted respectively to detect the localization of the fusion proteins. Lamin B1 for the

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nucleus and β-actin for the cytoplasm were used as cellular markers. N represents the nucleus and C represents the cytoplasm.

Fig.3. Nuclear import of BLM protein requires importin β1, RanGDP and NTF2. (A) Digitonin-permeabilized HeLa cells were incubated with GST-BLM642-1417-GFP in the presence of cytosol, importin β1, importin β1 plus RanGTP, RanGDP plus NTF2, importin β1 and RanGDP plus NTF2, or importin β1 and RanQ69LGTP plus NTF2. DAPI was used to

ACCEPTED MANUSCRIPT stain the nuclei. The green fluorescence was observed under fluorescence microscope. Original magnification was 1×200. (B) Digitonin-permeabilized HeLa cells were incubated with GST-NLSBLM-GFP or GST-OGFr-GFP in the presence of cytosol, importin β1, RanGDP

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plus NTF2, importin β1 and RanGDP plus NTF2. DAPI was used to stain the nuclei. The green fluorescence was observed under fluorescence microscope. Original magnification was 1×200. (C) GST-BLM642-1417 or GST immobilized on Glutathione-Sepharose beads were

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incubated with His-importin β1 (lanes 1 and 4), RanGDP plus NTF2 (lanes 2 and 5),

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His-importin β1 and RanGDP plus NTF2 (lanes 3 and 6) in transport buffer. After incubation, the bound proteins were eluted and analyzed by SDS-PAGE followed by Coomassie blue staining.

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Fig.4. BLM protein interacts with importin β1 in vivo and in vitro.

HEK-293T cells were transfected with plasmid pEGFP-BLM642-1417. Cells were lysed at 36 h post-transfection, and a co-immunoprecipitation assay was performed using either

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anti-importin β1 (A) or anti-GFP (B) antibodies. Immunoprecipitated proteins were detected

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by Western blotting using anti-GFP (A) or anti-importin β1 (B) antibody. (C) GST-BLM642-1417 or GST protein immobilized on Gluthatione-Sepharose beads were incubated with purified His-importin β1 for 2 h at 4℃. After washing, the bound proteins were separated by SDS-PAGE and immunoblotted for importin β1 using anti-importin β1 antibody. (D) His*Bind Resin-binded His-importin β1 protein and purified GST-BLM642-1417 were incubated for 2 h at 4℃. After washing, the bound proteins were separated by SDS-PAGE and immunoblotted for BLM642-1417 using anti-GST antibody.

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Fig.5. The NLS domain of BLM interacts with 14-16 HEAT repeats of importin β1. (A)

His*Bind

Resin-binded

His-importin

β1 protein

pEGFP-BLM642-1417

and

or

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pEGFP-BLM642-1417/NLSm transfected HEK-293T cell lysates were incubated for 2 h at 4℃. The complex was washed and then separated by SDS-PAGE and immunoblotted for EGFP-tagged proteins using anti-GFP antibody. The input samples were shown in lower panel.

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(B) GST-BLM642-1417 immobilized on Gluthatione-Sepharose beads were incubated with

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exogenous Myc-importin β1 truncations derived from plasmids-transfected HEK-293T cells for 2 h at 4℃. After washing, the bound proteins were separated by SDS-PAGE and immunoblotted for Myc-importin β1 truncations using anti-Myc antibody. The input samples

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were shown in lower panel.

Fig.6. Importin β1, Ran or NTF2 depletion disrupts the nuclear import of BLM. (A) HEK-293T cells were transfected with human importin β1 siRNA, Ran siRNA, NTF2

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siRNA and control siRNA, respectively. After 48 h transfection, cell lystaes were prepared

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and examined by Western blot analysis. Endogenous β-actin expression was used as internal controls. (B) HEK-293T cells were co-transfected with pEGFP-BLM642-1417, pEGFP-OGFr or pEGFP-C1 and the indicated siRNA. Forty-eight hours after transfection, subcellular localization of the fusion protein EGFP-BLM642-1417, EGFP-OGFr or EGFP alone was checked by fluorescence microscopy. Cells were counterstained with DAPI to detect the nuclei. In parallel, the nuclear and cytoplasmic fractions from cells transfected with the indicated plasmid and siRNA were extracted respectively to detect the fusion proteins. Lamin

ACCEPTED MANUSCRIPT B1 for the nucleus and β-actin for the cytoplasm were used as cellular markers. N represents the nucleus and C represents the cytoplasm. (C) The cytoplasmic localization of EGFP-BLM642-1417, EGFP-OGFr or EGFP is indicated as a percentage of the total number of

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cells counted. Approximately 200 cells were counted per experiment condition using an

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unbiased method. Data represent the mean ± SD of at least three independent experiments.

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Fig. 6

ACCEPTED MANUSCRIPT Nuclear import of BLM protein was reconstituted using importin β1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. BLM protein had direct binding to importin β1 through its NLS domain with the 14-16

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HEAT repeats of importin β1. siRNA-mediated knockdown of importin β1, Ran or NTF2 all reduced the nuclear accumulation of BLM protein in different degrees.

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These data demonstrate for the first time the nuclear trafficking mechanism of a DNA

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helicase.