Tip110, a novel interactor of Fe65, localizes to nucleus

Since Tip60 is not pro-apoptotic in cerebellar granule neurons, we tried to identify another potential player in RNF157/Fe65 pathway. Since nuclear Fe65 potently induced neuronal apoptosis in neurons, I reasoned that the proteins interacting with nuclear Fe65 could have an impact on neuronal death. A yeast-two hybrid screen was therefore conducted by A. Matz using a human fetal brain cDNA library to search for nuclear interactors of Fe65. Here, we identified Tip110, a nuclear RNA-binding protein (Gu et al., 1998), as a potential interactor of Fe65.

Tip110 is an HIV-1 Tat interacting protein (Liu et al., 2002) and has been implicated in RNA processing (Song et al., 2010), hematopoiesis (Liu et al., 2011), regulation of embryonic stem cells (Liu et al., 2012) as well as HIV-1-mediated gene transcription initiation and elongation (Liu et al., 2002; Zhao et al., 2014). However, the role of Tip110 in neuronal survival has not yet been investigated.

Therefore, I first confirmed the interaction of Fe65 and Tip110 in HEK293T cells (Fig 3.17a). HEK293T cells were transfected with either the HA-Tip110 plasmid or the Flag-Fe65 construct, or both of the HA-Tip110 and the Flag-Fe65 plasmids. Cell lysates were subjected to coimmunoprecipitation with the Flag antibody and immunoblotting with the HA antibody. I found that Tip110 interacted with Fe65. Next, I asked if Tip110 localizes to nucleus also in neurons. Due to the lack of a specific Tip110 antibody, I overexpressed HA-tagged Tip110 in

hippocampal neurons and observed an expected nuclear localization (Fig 3.17b), as already shown in HeLa cells (Gu et al., 1998).

3.3.3.1 Tip110 promotes neuronal apoptosis

Having established the interaction and localization of Tip110, I further asked if Tip110 has a role in mediating neuronal apoptosis. First of all, we conducted the gain-of-function analyses in cerebellar granule neurons as well as in the hippocampal neurons. Here, Tip110 was overexpressed together with β-Gal plasmid in the cultured neurons at DIV2. Neurons were fixed and analyzed at DIV6. Interestingly, overexpression of Tip110 induced cell death up to 2 folds as compared to the control cerebellar granule neurons (Fig 3.18a). In hippocampal neurons, Tip110 induced an even stronger apoptotic phenotype (Fig 3.18b, c). These results indicated that Tip110 acts in a pro-apoptotic manner in neurons.

a

b

Fig 3.17 Tip110 interacts with Fe65 and localizes to the nucleus.

a) Lysates of HEK293T cells, transfected with control vector, together with the Flag-Fe65 or the HA-Tip110 plasmid or with both of the Flag-Fe65 and the HA-Tip110 plasmids, were immunoprecipitated with the Flag antibody and subjected to immunoblotting with the myc antibody.

b) Hippocampal neurons were transfected with the HA-Tip110 plasmid and subjected to immunohistochemistry with the HA antibody, the neuronal marker TuJ1 and the DNA dye DAPI. Scale bar = 20 µm.

Results

3.3.3.2 Tip110 acts together with Fe65 and RNF157 in controlling neuronal survival To establish Tip110 as a component in the RNF157/Fe65 pathway, I first generated Tip110 RNAi constructs and verified their efficacy in HEK293T cells expressing the HA-Tip110 plasmid (Fig 3.19a). Among the four RNAi constructs only Tip110 RNAi #3 was efficient in knocking down Tip110 and was used in the following experiments.

To investigate if Fe65 regulates neuronal apoptosis together with Tip110, I carried out epistasis experiments. Here, I overexpressed Fe65, knocked down Tip110, or expressed both the Fe65 expression plasmid and Tip110 RNAi plasmid together with the β-Gal plasmid in hippocampal neurons at DIV2. After 5 days, hippocampal neurons were immunostained with the β-Gal antibody and the DNA dye DAPI and analyzed. Interestingly, I found that while knockdown of Tip110 did not show any effect on neurons, it neutralized the apoptosis induced by Fe65 (Fig 3.19b, c). These experiments suggested that Tip110 is required for nuclear Fe65 to induce apoptosis. In further epistasis analysis, I examined if Tip110 knockdown could rescue the strong apoptosis induced by RNF157 knockdown. Here, I knocked down Tip110, RNF157 or both Tip110 and RNF157 together with overexpression of the β-Gal plasmid in hippocampal

a b c

Fig 3.18 Tip110 induces neuronal apoptosis in cerebellar granule and in hippocampal neurons.

a) Cerebellar granule neurons (CGNs) transfected at DIV2 with the empty vector or the Tip110 expression plasmid together with the β-Gal plasmid were subjected to immunocytochemistry with the β-Gal antibody and the DNA dye bisbenzimide at DIV6. A total of 541 neurons from 3 independent experiments were analyzed (Student’s t-test, ***p<0.001, mean +s.e.m.). Courtesy of A. Matz.

b) Hippocampal neurons were transfected at DIV2 with control plasmid or the Tip110 expression plasmid together with the β-Gal plasmid and analyzed at DIV6. A total of 429 neurons from 3 independent experiments were included in the analysis (Student’s t-test, *** p<0.001, mean +s.e.m.).

c) Representative images of neurons in 3.8b. Arrowhead indicates the nucleus of transfected neurons. Insets depict higher magnification of nuclei and the arrow indicates the pyknotic nucleus.

neurons at DIV2. Neurons were subjected to immunochemistry and analyzed at DIV7. I found that knockdown of Tip110 led to a partial rescue of the RNF157 RNAi-induced cell death (Fig 3.19d). These analyses indicated that Tip110 is a component of the RNF157/Fe65 pathway.

a b c

d

Fig 3.19 Knockdown of Tip110 rescued neuronal apoptosis induced by Fe65 and ameliorated the cell death upon RNF157 knockdown in HPNs.

a) Lysates of HEK293T cells, transfected with the HA-Tip110 plasmid together with empty vector and different Tip110 RNAi constructs, were subjected to immunoblotting with the HA and the 14-3-3β antibodies. The latter was a loading control.

b) Hippocampal neurons were transfected at DIV2 with control vectors, the Fe65 plasmid, the Tip110 RNAi construct or both the Fe65 expression and the Tip110 RNAi plasmids and fixed at DIV7.

A total of 1447 neurons in 4 independent experiments were analyzed (ANOVA, *** p<0.001, mean +s.e.m.).

c) Representative images of neurons in 3.19b. Insets depict higher magnification of nuclei. Scale bar = 20 μm.

d) Hippocampal neurons were transfected at DIV2 with control vectors, the Tip110 RNAi plasmid, the RNF157 RNAi plasmid or both the Tip110 RNAi and the RNF157 RNAi plasmids and fixed at DIV7. A total of 1410 neurons in 4 independent experiments were included in the analysis (ANOVA, ** p<0.01, *** p<0.001, mean +s.e.m.).

Results