The kinesin motor KIF1C is a putative transporter of the exon junction complex in neuronal cells - PubMed (original) (raw)
The kinesin motor KIF1C is a putative transporter of the exon junction complex in neuronal cells
Maike Nagel et al. RNA. 2022.
Abstract
Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons.
Keywords: RNA transport; exon juncion complex; kinesin.
Published by Cold Spring Harbor Laboratory Press for the RNA Society.
Figures
FIGURE 1.
Interaction of exon junction complex components and KIF1C. (A) Protein–protein interaction network of KIF1C: KIF1C interactors identified in this study are indicated in red; additional proteins associated with the exon junction complex (according to GO terms) are depicted in gray. (B) Immunoprecipitation of KIF1C complexes in differentiated SH-SY5Y cells stably overexpressing KIF1C-HA constructs. Interaction with new interaction partners identified by MS was confirmed in the KIF1C IPs; as expected, these proteins do not interact with the HA-tag and wild-type KIF5A-HA (control). (C) Coimmunoprecipitation of eIF4A3 in differentiated SH-SY5Y cell lines. KIF1CWT and KIF1CG102A were detected in the Co-IP, while KIF5A-HA (control) was missing. All other tested proteins are known to interact with eIF4A3 and were accordingly identified in all IP samples. The control Co-IP with an IgG antibody showed no signal for any of the tested proteins.
FIGURE 2.
Colocalization of KIF1C and EJC components eIF4A3 and RBM8A. Images show the colocalization of KIF1CWT and the EJC components at the tips of cellular processes. In contrast, KIF1CG102A and the EJC components colocalize in the pericentrosomal region. Due to lack of a suitable antibody reliably detecting endogenous MAGOH in neuronal cells, staining for MAGOH is not reported here. The scale bars represent 10 µm.
FIGURE 3.
RNA-mediated interaction of KIF1C and the EJC. (A) RNase IP was performed in differentiated SH-SY5Y cells overexpressing HA-tagged KIF1CWT or KIF1CG102A. The proteins interact in a normal IP with KIF1C and the interaction is lost when the samples are treated with RNase I before the IP, whereas an RNase inhibition can restore the interaction. (B) Complex Capture (2C) in HEK 293 cells overexpressing HA-tagged KIF1CWT or KIF1CG102A. The input shows signal in cross-linked (Cl) and not cross-linked (NoCl) samples, whereas in the 2C samples only proteins cross-linked to RNA are detected. Histon H3 is a DNA-binding protein and used as a control to confirm the specificity of the method for RNA–protein interactions. As a positive control, the known RNA-binding protein hnRNP C1/C2 was used. It is detected in the 2C cross-linked samples as well as wild-type and mutant KIF1C.
Maike Nagel
References
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