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«Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the Integrator complex a a b b c Jeanne N. Jodoin, Poojitha ...»

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Nuclear-localized Asunder regulates cytoplasmic dynein localization via

its role in the Integrator complex

a a b b c

Jeanne N. Jodoin, Poojitha Sitaram, Todd R. Albrecht, Sarah B. May, Mohammad Shboul,

a c,d b,1 a,1

Ethan Lee, Bruno Reversade, Eric J. Wagner, and Laura A. Lee

a

Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA b Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, TX 77030, USA c Institute of Medical Biology, A*STAR, Singapore, Singapore 138648 d Department of Pediatrics, National University of Singapore, Singapore 119228

Co-corresponding authors. Address correspondence to:

Laura Lee (laura.a.lee@vanderbilt.edu) or Eric Wagner (eric.j.wagner@uth.tmc.edu) RUNNING HEAD: INT regulates dynein localization

ABBREVIATIONS

ASUN, Asunder BICD2, Bicaudal D2 CDK1, Cyclin-dependent kinase 1 CENP-F, Centromere protein F CHY, mCherry CPSF30, Cleavage Polyadenylation Specificity Factor 30 DHC, dynein heavy chain DIC, dynein intermediate chain dASUN, Drosophila Asunder GFP, green fluorescent protein hASUN, human Asunder INT, Integrator complex IntS, Integrator subunit mASUN, mouse Asunder NT, non-targeting NE, nuclear envelope NEBD, nuclear envelope breakdown NLS, nuclear localization sequence siRNA, small-interfering RNA snRNA, small nuclear RNA ABSTRACT We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus-centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, though the mechanisms underlying this regulation remain unknown. We have also recently identified ASUN as a functional component of Integrator (INT), a multi-subunit complex required for 3’-end

Supplemental Material can be found at:

http://www.molbiolcell.org/content/suppl/2013/07/29/mbc.E13-05-0254.DC1.html processing of small nuclear RNAs (snRNAs). We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE.

Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN-siRNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence (NLS) blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. Additionally, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support amodel in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.

INTRODUCTION

Dynein, a minus-end-directed molecular motor, is a large multimeric complex that can be divided into distinct regions (Holzbaur and Vallee, 1994; Kardon and Vale, 2009). Protruding from the head region are two microtubule-binding domains that allow the motor to walk processively along the microtubule towards its minus end. This movement is driven by the forcegenerating ATPase activity of the catalytic domains found within the head region of the motor.

The stem region, consisting of multiple light, light intermediate, and intermediate chains, is the most variable and is widely considered to serve as the binding site for dynein adaptors.

Within the cell, dynein exists in association with its activating complex, dynactin (Schroer, 2004). Together, the dynein-dynactin complex performs diverse functions within the cell, ranging from cargo transport, centrosome assembly, and organelle positioning to roles in chromosome alignment and spindle positioning during mitosis (Holzbaur and Vallee, 1994;

Kardon and Vale, 2009). Dynein-dynactin complexes are subject to multiple layers of regulation, including binding of accessory proteins, phosphorylation, subunit composition, and subcellular localization (Kardon and Vale, 2009). Localized pools of dynein have been identified and shown to be required for critical processes in the cell, although the mechanisms underlying the control of dynein localization are poorly understood (Kardon and Vale, 2009).

Across phyla, a stably anchored subpopulation of dynein has been reported to exist on the NE of cells (Gonczy et al., 1999; Robinson et al., 1999; Salina et al., 2002; Payne et al., 2003;

Anderson et al., 2009). This pool of dynein is required for both stable attachment of centrosomes to the NE prior to nuclear envelope breakdown (NEBD) and migration of centrosomes to opposite sides of the nucleus, thereby ensuring proper positioning of the bipolar spindle (Vaisberg et al., 1993; Gonczy et al., 1999; Robinson et al., 1999; Malone et al., 2003; Anderson et al., 2009; Splinter et al., 2010; Bolhy et al., 2011; Jodoin et al., 2012; Sitaram et al., 2012).

Additionally, this pool of dynein appears to facilitate NEBD by forcibly tearing the NE, although the precise mechanism remains unknown (Beaudouin et al., 2002; Salina et al., 2002).

Two proteins, Bicaudal D2 (BICD2) and Centromere protein F (CENP-F), have been shown to directly anchor dynein to the nuclear surface at the G2/M transition in HeLa cells (Splinter et al., 2010; Bolhy et al., 2011). BICD2, a dynein adaptor protein, directly binds dynein and nucleoporin RanBP2, thereby anchoring the motors to the NE (Splinter et al., 2010). CENPF directly interacts with dynein adaptor proteins NudE/EL and nucleoporin Nup133 to effectively anchor dynein to the NE (Bolhy et al., 2011). In both Drosophila spermatocytes and cultured human cells, we previously identified ASUN as an additional regulator of dynein recruitment to the NE at G2/M of meiosis and mitosis, respectively, although physical interaction between ASUN and dynein has not been demonstrated (Anderson et al., 2009; Jodoin et al., 2012).





Spermatocytes within the testes of Drosophila asun males arrest at prophase of meiosis I with a severely reduced pool of perinuclear dynein and centrosomes that are not attached to the nuclear surface (hence the name “asunder”) (Anderson et al., 2009). Spermatocytes that progress beyond this arrest exhibit defects in spindle assembly, chromosome segregation, and cytokinesis during the meiotic divisions. Using cultured human cells, we found that small-interfering RNA (siRNA)-mediated down-regulation of the human homologue of ASUN (hASUN) similarly resulted in reduction of perinuclear dynein during prophase of mitosis (Jodoin et al., 2012).

Additional defects observed following loss of hASUN included nucleus-centrosome uncoupling, abnormal mitotic spindles, and impaired progression through mitosis.

In either Drosophila or cultured human cells, a direct mechanism for promotion of perinuclear dynein by ASUN has not been elucidated, although localization changes in ASUN coincide with the accumulation of dynein on the NE. Drosophila ASUN (dASUN) is largely restricted within the nucleus of early G2 spermatocytes and first appears in the cytoplasm during late G2, roughly coincident with the initiation of dynein recruitment to the nuclear surface (Anderson et al., 2009). Similarly, in prophase HeLa cells, when a perinuclear pool of dynein forms transiently at G2/M, hASUN is diffusely present in the cytoplasm (Jodoin et al., 2012).

Based on these temporal associations of the localizations of ASUN and perinuclear dynein, we previously proposed that the cytoplasmic pool of ASUN likely mediates recruitment of dynein motors to the NE (Anderson et al., 2009; Jodoin et al., 2012).

INT is an evolutionarily conserved complex consisting of 14 subunits, although its biology is poorly understood (reviewed in Chen and Wagner, 2010). INT was originally identified due to its association with the C-terminal tail of RNA polymerase II and was subsequently shown to be required for 3’-end processing of snRNAs (Baillat et al., 2005). These processed snRNAs play roles in gene expression via intron removal and further processing of pre-mRNAs (Matera et al., 2007). To discover novel components of INT that are required for its snRNA-processing function, a cell-based assay was developed in which generation of a GFP signal due to incomplete processing of a reporter U7 snRNA served as a read-out of INT activity (Chen et al., 2012). Using this approach, dASUN was identified as a functional component of INT: down-regulation of dASUN led to increase misprocessing of U7 and spliceosomal snRNA, thereby indicating its requirement for activity of the complex (Chen et al., 2012). Furthermore, dASUN was shown to biochemically interact with INT in a stoichiometric manner, an association that it is conserved in humans (Malovannaya et al., 2010; Chen et al., 2012).

Collectively, these data provide compelling evidence that ASUN is a core Integrator subunit.

Given the divergent nature of the known activities of ASUN – critical regulator of cytoplasmic dynein localization and essential component of a nuclear snRNA-processing complex – we sought herein to determine whether these roles are independent of each other or derived from a common function. We find that depletion of individual INT components from HeLa cells results in loss of perinuclear dynein, recapitulating the phenotype observed in hASUN-siRNA cells (Jodoin et al., 2012). Additionally, we find that forced localization of either hASUN or dASUN to the cytoplasm inhibits their capacity to recruit dynein to the NE in the absence of endogenous ASUN. We present a model in which ASUN acts within the nucleus in concert with other subunits of the Integrator complex, likely via processing of a critical RNA target(s), to promote recruitment of cytoplasmic dynein motors to the NE at G2/M.

RESULTS Multiple INT subunits are required for dynein recruitment to the NE Two ASUN-dependent cellular functions have been reported: dynein recruitment to the NE at G2/M, and proper processing of snRNA by INT (Chen et al., 2012; Jodoin et al., 2012).

We hypothesized that other components of the INT complex, like hASUN, may be required to promote dynein recruitment to the NE. To test this hypothesis, we performed siRNA-mediated knockdown of individual INT subunits in HeLa cells and assessed dynein localization. Prior to fixation and immunostaining for dynein intermediate chain (DIC), siRNA-treated cells were incubated briefly with 5 µM nocodazole to stimulate recruitment of dynein-dynactin complexes to the NE. This treatment has been documented to recapitulate, in non-G1 cells, the enrichment of functional dynein-dynactin complexes on the NE that normally occurs at G2/M, making this an ideal assay for identifying factors involved in dynein localization (Beswick et al., 2006;

Hebbar et al., 2008; Splinter et al., 2010; Bolhy et al., 2011; Jodoin et al., 2012).

Consistent with our previous report, we found that 78% of non-targeting (NT) control siRNA cells had a striking enrichment of dynein on the NE after brief nocodazole treatment (Figure 1, A and O); in contrast, the percentage of cells with perinuclear dynein was reduced to 20% following hASUN depletion (Figure 1, C and O) (Jodoin et al., 2012). In most cases, we found that treatment of cells with siRNA targeting individual INT subunits (each designated “IntS” followed by a unique identifying number) resulted in a similar reduction of cells with perinuclear dynein (Figure 1, D-I, K, M, and N). Importantly, we did not observe any overt effect on cellular health or growth after treatment with INT-targeting siRNAs, arguing against any potential reduction in cellular fitness as the cause of reduced perinuclear dynein. To further quantify the dynein phenotype, we compared the DIC immunofluorescence signals on the NE to that of the cytoplasm and also determined the average peak DIC intensity on the NE for each knockdown as previously described (Figure S1) (Jodoin et al., 2012). Depletion of IntS1-6, 9, 11, or 12 resulted in a marked decrease in both the ratio of NE-to-cytoplasmic dynein and the peak intensity of DIC on the NE, comparable to that observed for hASUN. IntS7 and IntS10 were the two exceptions: depletion of either of these INT subunits had no effect on perinuclear dynein accumulation compared to control cells (Figure 1, J, L, and O; Figure S1). We confirmed that all targeted proteins were efficiently depleted by immunoblotting of cell lysates after siRNA treatment or by a second, non-overlapping, siRNA (Figure S2). Taken together, these data show that the majority of the individual INT subunits are required for dynein recruitment to the NE.

We considered the possibility that loss of dynein accumulation on the NE upon INT depletion could be secondary to cell-cycle arrest. We performed fluorescence-activated cell sorting (FACS) analysis of DNA-stained HeLa cells following knockdown of individual INT subunits (Figure S3). We observed no differences between the cell-cycle profile of hASUN- or other INT subunit-siRNA cells and that of control NT-siRNA cells (Figure S3A). We previously reported that hASUN depletion from HeLa cells results in a slightly increased mitotic index (Jodoin et al., 2012); we show herein that depletion of other INT subunits has a similar effect (Figure S3B). We also found that the percentage of prophase cells, the stage at which dynein normally accumulates on the NE, is slightly increased upon knockdown of ASUN or other INT subunits from HeLa cells (Figure S3C). These results indicate that loss of dynein recruitment to the NE in cells depleted of INT is not due to any substantial cell-cycle perturbation.

Table 1 summarizes our observations of the requirements for INT subunits in dynein localization and the previously reported requirements for INT subunits in snRNA processing (Ezzeddine et al., 2011; Chen et al., 2012). The two data sets compare favorably in that, for both processes, most INT subunits are required, whereas IntS10 is expendable. IntS7, however, was the sole exception in that it was shown to be required for snRNA processing, yet we found no effect of its down-regulation on dynein recruitment to the NE (Chen et al., 2012). Overall, these data are consistent with a model in which hASUN regulates dynein localization in an INT complex-dependent manner.



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