Nuclear speckle integrity and function require TAO2 kinase

Abstract

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.

Keywords: TAOK2; mRNA export; nuclear speckles; splicing.

Fig. 1.

TAO2 is localized at nuclear speckles and maintains the basal levels of nuclear speckle factors. (A) A549 cells were subjected to immunofluorescence microscopy with rabbit anti-TAO2 serum and mouse SC35 antibody. The enlarged image on the right depicts a nuclear speckle from the merge panel (marked region). (Scale bars, 5 μm.) n = 3. (B) A549 cells or A549 cells with the SON gene tagged with Flag, HA, and AID were transfected with control siRNA or with an siRNA pool targeting TAO2 and then subjected to immunofluorescence microscopy with antibodies against TAO2 and SC35, or with anti-FLAG antibodies to detect SON protein. White arrows indicate cells with decreased levels of TAO2 compared with neighboring cells. (Scale bars, 5 μm.) (C) A549 cells were transfected with control siRNA or siRNAs targeting TAO2, and whole-cell lysates were subjected to Western blot to detect endogenous TAO2, SC35, SON, nucleolin, and hnRNP K protein levels. β-actin serves as loading control. n = 3. The intensity of each protein band was quantified and normalized to β-actin levels.

Fig. 2.

Nuclear speckle proteome is associated with TAO2. Nuclear lysates from A549 cells were subjected to immunoprecipitation with control rabbit IgG or TAO2 antibody. (A) TAO2-interacting proteins were identified by mass spectrometry. The heatmap shows relative abundances of selected hits representing proteins that overlap with constituents of nuclear speckles. Control immunoprecipitation (IP) with IgG is compared with immunoprecipitation performed with TAO2 antibody. NA represents hits not detected in IgG-immunoprecipitated samples. (B) Immunoprecipitates were probed with the indicated antibodies to confirm the interactions between TAO2 and selected hits in A. NXF1 is used as negative control since it did not interact with TAO2 in the mass spectrometry analysis. n = 3. (C) A549 cells were transfected with control siRNA or siRNAs targeting TAO2, and whole-cell lysates were subjected to Western blot to detect endogenous TAO2, SRSF1, and Aly/Ref. β-actin serves as loading control. n = 3.

Fig. 3.

Pool of the TAO2–SRSF1 complex is localized at the periphery of nuclear speckles. (A) A549 cells grown on coverslips were fixed and subjected to PLAs with the indicated primary antibodies. (Scale bars, 5 μm.) (B) PLA signals were quantified in 100 cells for each condition. Values are means ± SD. n = 3. (C and D) A549 cells were subjected to PLA to detect the TAO2–SRSF1 complex followed by immunofluorescence microscopy to stain nuclear speckles with an antibody against SC35. n = 3. (C) Two-dimensional images of PLA signals merged with nuclear speckles and DNA. (Scale bars, 2 μm.) (D) Three-dimensional projection of PLA signals and nuclear speckles. (I) Low magnification of the maximum-intensity projection (MIP) is shown. PLA signal of the TAO2–SRSF1 complex (red), nuclear speckles (SC35, green), and chromatin (blue). (II and III) High magnification of the MIP surfaces is shown for PLA signal of the TAO2–SRSF1 complex (red) and nuclear speckles are stained in green. (Scale bars, 2 μm.) (IV) Pairwise merged surface created for PLA signal of the TAO2–SRSF1 complex (red) and nuclear speckles (green) is shown. (V–VII) Views of a chromatin merged surface created for (V) PLA signal of the TAO2–SRSF1 complex (red), (VI) nuclear speckles (green), and (VII) PLA signal of TAO2–SRSF1 (red) and nuclear speckles (green) are shown. (Scale bars, 2 μm.) The squared marked area in VII is enlarged and shown in the view of 3D isometric MIP-rendering volume (Top) or 3D isometric blend-rendering volume (Bottom). Both 3D rendered volumes are presented in 90° alternative views to provide visual and spatial emphasis for the colocalization (yellow) of the TAO2–SRSF1 complex (red) and nuclear speckles (green). To calculate the percentage of the TAO2–SRSF1 complex colocalization with nuclear speckles, 156 cells were analyzed and 42% of the PLA signal was found at nuclear speckles.

Fig. 4.

Partial depletion of TAO2 impairs splicing and nuclear export of influenza virus M mRNA. (A) Schematic representation of influenza virus M1 and M2 mRNAs. Boxes denote exons and the line denotes an intron. (B–F) A549 cells were treated with control siRNA or siRNAs targeting TAO2 followed by infection with WSN at an MOI of 2 for 8 h. (B) Purified RNA from total cell lysates was subjected to qPCR to measure TAO2 mRNA. (C) Total cell lysates were subjected to Western blot to detect TAO2 protein levels. β-actin serves as loading control. n = 3. (D) Purified RNA from total cell lysates was subjected to qPCR to measure M1, M2, NS1, and NS2 mRNA levels. (E and F) The ratios of M2/M1 (E) and NS2/NS1 (F) mRNA levels are shown. Graphs show mean ± SD. n = 3. *P < 0.05, **P < 0.01. (G) A549 cells were treated with control or siRNAs targeting TAO2 followed by infection with WSN at an MOI of 2 for 8 h. smRNA-FISH was performed to detect M mRNA. (H) Nuclear-to-cytoplasmic fluorescence intensity (N/C ratio) was quantified. Control, n = 177 cells; siTAO2, n = 181 cells. **P < 0.01.

Fig. 5.

Partial depletion of TAO2 results in inhibition of influenza virus replication and alters the levels of a subset of cellular mRNAs. (A) A549 cells were transfected with control siRNA or siRNAs targeting TAO2 followed by infection with WSN at an MOI of 2 for 8 h. Total cell lysates were subjected to Western blot to detect the levels of β-actin (loading control), TAO2, and viral proteins HA, NP, M1, and M2. (B and C) Cells were infected with WSN at an MOI of 0.01 and supernatants and cell lysates were collected at the indicated time points after infection. (B) Supernatants were used in viral replication assays following NP expression. (C) Cell lysates were subjected to Western blot analysis to assess the levels of TAO2, SON, and β-actin (loading control). (D) Depletion of TAO2 alters the levels of a subset of cellular mRNAs. Total mRNA from cells treated with control siRNA or siRNAs targeting TAO2 was subjected to RNA-seq analysis. The volcano plot depicts total levels of individual cellular mRNAs upon TAO2 knockdown. NS, not signficantly changed; FC, fold change. (D, Inset) Selected interferon-regulated mRNAs whose levels were up-regulated upon TAO2 knockdown, measured by qPCR. Graphs show means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3. (E) Gene set enrichment analysis of the KEGG database shows enrichment in pathways involved in the regulation of viral infections upon TAO2 depletion.