Heterogeneous Nuclear Ribonucleoprotein K, an RNA-Binding Protein, Is Required for Optic Axon Regeneration inXenopus laevis

Abstract

Axotomized optic axons of Xenopus laevis, in contrast to those of mammals, retain their ability to regenerate throughout life. To better understand the molecular basis for this successful regeneration, we focused on the role of an RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) K, because it is required for axonogenesis during development and because several of its RNA targets are under strong post-transcriptional control during regeneration. At 11 d after optic nerve crush, hnRNP K underwent significant translocation into the nucleus of retinal ganglion cells (RGCs), indicating that the protein became activated during regeneration. To suppress its expression, we intravitreously injected an antisense Vivo-Morpholino oligonucleotide targeting hnRNP K. In uninjured eyes, it efficiently knocked down hnRNP K expression in only the RGCs, without inducing either an axotomy response or axon degeneration. After optic nerve crush, staining for multiple markers of regenerating axons showed no regrowth of axons beyond the lesion site with hnRNP K knockdown. RGCs nonetheless responded to the injury by increasing expression of multiple growthassociated RNAs and experienced no additional neurodegeneration above that normally seen with optic nerve injury. At the molecular level, hnRNP K knockdown during regeneration inhibited protein, but not mRNA, expression of several known hnRNP K RNA targets (NF-M, GAP-43) by compromising their efficient nuclear transport and disrupting their loading onto polysomes for translation. Our study therefore provides evidence of a novel post-transcriptional regulatory pathway orchestrated by hnRNP K that is essential for successful CNS axon regeneration.

c-Jun N-Terminal Kinase Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein K Regulates Vertebrate Axon Outgrowth via a Posttranscriptional Mechanism

“…For example, in developing neurons of Xenopus laevis, heterogeneous nuclear ribonucleoprotein K (hnRNP K), an RNA-binding protein, is involved in nuclear export and translational control of multiple cytoskeletal-related transcripts needed for axonogenesis [e.g., type IV neurofilaments (NFs), actin-related protein 2 (ARP2), growth-associated protein 43 (GAP43), and microtubule-associated protein tau]; knockdown of hnRNP K by antisense morpholino oligonucleotide (MO) in Xenopus leads not only to the loss of protein as a result of the inefficient nuclear export and ineffectual loading of these hnRNP K-targeted mRNAs with polyribosomes for translation but also the failure of both embryonic axon initiation and optic axon regeneration (Liu et al, 2008(Liu et al, , 2012Liu and Szaro, 2011).…”

“…Single-label immunofluorescence in culture was performed as described previously (Undamatla and Szaro, 2001), using TRITC-conjugated avidin D (1:1000; Vector Laboratories). Double-label immunofluorescence in culture was performed as described previously (Liu and Szaro, 2011). For all cultures, nuclei were stained with 4Ј,6-diamidino-2-phenylindole (DAPI; Invitrogen).…”

“…RNA-binding protein/RNA coimmunoprecipitation. Coimmunoprecipitation of mRNP complexes was performed as described, with minor modifications (Liu et al, 2008). Lysates from stage 37/38, RNA-injected embryos (30 pooled per sample) were incubated with protein G-agarose beads (P7700; Sigma-Aldrich) coated with anti-GFP (Rockland) for 2 h at room temperature.…”

“…qRT-PCR was performed as described previously, with minor modifications (Liu et al, 2008;Liu and Szaro, 2011). For culture, cDNA was synthesized from 5 g of total RNA using SuperScript III reverse transcriptase (Invitrogen) and oligo-dT 12-18 for priming, and qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems), 1 l of cDNA template, and 150 nM each forward and reverse primers.…”

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

“…The time course and spatial distribution of regenerating axons after optic nerve injury is well-characterized. Tectal reinnervation of frog RGCs starts from about 6 weeks after axotomy and RGC axons restore a topographic visual projection into the optic tectum, which coincides in most species with recovery of normal visual responses (Maturana et al 1959;Singman and Scalia 1991;Dunlop et al 1997;Dunlop 2003;Liu et al 2012).…”