Nephronophthisis gene products display RNA-binding properties and are recruited to stress granules

Abstract

Mutations of cilia-associated molecules cause multiple developmental defects that are collectively termed ciliopathies. However, several ciliary proteins, involved in gating access to the cilium, also assume localizations at other cellular sites including the nucleus, where they participate in DNA damage responses to maintain tissue integrity. Molecular insight into how these molecules execute such diverse functions remains limited. A mass spectrometry screen for ANKS6-interacting proteins suggested an involvement of ANKS6 in RNA processing and/or binding. Comparing the RNA-binding properties of the known RNA-binding protein BICC1 with the three ankyrin-repeat proteins ANKS3, ANKS6 (NPHP16) and INVERSIN (NPHP2) confirmed that certain nephronophthisis (NPH) family members can interact with RNA molecules. We also observed that BICC1 and INVERSIN associate with stress granules in response to translational inhibition. Furthermore, BICC1 recruits ANKS3 and ANKS6 into TIA-1-positive stress granules after exposure to hippuristanol. Our findings uncover a novel function of NPH family members, and provide further evidence that NPH family members together with BICC1 are involved in stress responses to maintain tissue and organ integrity.

Figure 1

RNA interaction with BICC1 and the ankyrin-repeat proteins ANKS3, ANKS6 and INVERSIN. (a) Venn diagram (www.bioinformatics.psb.urgent.be/webtools/Venn/), representing the overlap between RNA molecules interacting with BICC1, ANKS3, ANKS6 and INVERSIN. (b) The top two binding motifs for BICC1, INVERSIN, ANKS3, and ANKS6. Two putative binding motifs were also identified for ARL13B, albeit with much lower statistical significance. No significant binding motif (p < 0.05) was identified for BBS3.

Figure 2

RNA-binding properties of BICC1, ANKS3, ANKS6 and INVERSIN. (a) Depicted is the distribution of query regions with certain gene features. All four proteins recognize exons in transcripts. (b) Depicted is the distribution of query regions in the genome grouped by gene type. All four proteins recognize protein-coding regions. The analysis was performed with the RNA Centric Analysis System (RCAS) report tool on Galaxy (BIMSB, Max-Delbrück Center for Molecular Medicine, Berlin).

Figure 3

Interaction between BICC1 and AGO2 mRNA. (a) Integrative Genomics Viewer (IGV) snapshot of the FLASH experiments showing FLAG-tagged BICC1 (green) binding on AGO2 exon, the background control (grey) and the position of the PEAKachu results (orange). The coverage of uniquely mapped alignments for each profile is shown in the data range. (b) Flag-tagged BICC1 was precipitated, and interacting AGO2 mRNA was reverse-transcribed and amplified by PCR, yielding a 145 bp band. Controls without reverse transcriptase (RT) were essentially negative. In the bar graph, the input was adjusted to 100%. The RNA-precipitation assays were performed three times. The uncropped electrophoresis gel is shown in Supplementary Fig. 13. Alpha Imager V.4.1.0.2 was used for visualization.

Figure 4

Localization of ANKS3 in response to hippuristanol. (a) HeLa cells were transfected with RFP-tagged ANKS3, and either exposed to DMSO or 1 mM hippuristanol for 1 h. TIA-1-positive stress granules formed in response to 1 mM hippuristanol treatment. However, no co-localization was observed between TIA-1-positive stress granules and ANKS3. The experiment was performed twice, analyzing a total of 40 images and 14 z-stacks. (b) Co-transfection of GFP-tagged AGO2 or treatment with 1 mM hippuristanol did not alter the localization of ANKS3, while AGO2 partially co-localized with TIA-1-positive stress granules. The experiment was performed twice, analyzing a total of 41 images and 18 z-stacks.

Figure 5

Localization of ANKS6 in response to hippuristanol. (a) HeLa cells were transfected with RFP-tagged ANKS6, and either exposed to DMSO or 1 mM hippuristanol for 1 h. TIA-1-positive stress granules formed in response to 1 mM hippuristanol treatment. However, no co-localization was observed between TIA-1-positive stress granules and ANKS6. The experiment was performed three times, analyzing a total of 23 images and 12 z-stacks. (b) Co-transfection of GFP-tagged AGO2 (lower two panels) or treatment with 1 mM hippuristanol did not alter the localization of ANKS6, while AGO2 partially co-localized with TIA-1-positive stress granules. The experiment was performed three times, analyzing a total of 33 images and 15 z-stacks.

Figure 6

Localization of BICC1 in response to hippuristanol. (a) HeLa cells were transfected with RFP-tagged BICC1, and either exposed to DMSO or 1 mM hippuristanol for 1 h. BICC1 accumulated in granules without co-localization with TIA-1 in the absence of stress. Addition of hippuristanol recruited BICC1 into TIA-1-positive stress granules. The experiment was performed four times, analyzing a total of 58 images and 15 z-stacks. (b) Co-transfection of GFP-AGO2 caused partial co-localization of BICC1 and AGO2 with TIA-1-positive granules. Hippuristanol exposure recruited most BICC1/AGO2-granules into TIA-1-positive stress granules. The experiment was performed four times, analyzing a total of 91 images and 25 z-stacks.

Figure 7

Localization of INVERSIN in response to hippuristanol. (a) HeLa cells were transfected with RFP-tagged INVERSIN, and either exposed to DMSO or 1 mM hippuristanol for 1 h. INVERSIN assumed a diffuse cellular localization. Upon treatment with 1 mM hippuristanol, INVERSIN accumulated in TIA-1-positive stress granules. The experiment was performed four times, analyzing a total of 72 images and 22 z-stacks. (b) Co-transfection of GFP-tagged AGO2 did not alter the localization of INVERSIN. However, hippuristanol exposure recruited AGO2/INVERSIN-granules into TIA-1-positive stress granules. The experiment was performed five times, analyzing a total of 67 images and 24 z-stacks.

Figure 8

BICC1-mediated recruitment of NPH proteins into stress granules. (a) Co-expression of BICC1 recruited ANKS3 into granules that co-localized with TIA-1 in response to 1 mM hippuristanol. The experiment was performed two times, analyzing a total of 18 z-stacks. (b) Co-expression of BICC1 recruited ANKS6 into granules that co-localized with TIA-1 in response to 1 mM hippuristanol. The experiment was performed two times, analyzing a total of 17 z-stacks. (c) GFP-tagged INVERSIN and RFP-tagged BICC1 co-localized in granules that overlapped with TIA-1-positive stress granules even in the absence of hippuristanol. Hippuristanol treatment (1 mM for 1 h) increased the number of TIA-1-positive stress granules that largely co-localized with INVERSIN and BICC1. In this experiment, a total of 19 z-stacks were analyzed.