Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnpr\(^{tm1a/tm1a}\)) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnpr\(^{tm1a/tm1a}\) mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with gamma-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.

Axon growth, a fundamental process of neuron development, is regulated by both intrinsic and external guidance signals. Impairment of axon growth and maintenance is implicated in the pathogenesis of neurodegenerative disorders such as Amyotrophic Lateral Sclerosis and Alzheimer’s disease (AD). Axon growth is driven by several post-transcriptional RNA processing mechanisms, including alternative splicing, polyadenylation, subcellular localization, and translation. These mechanisms are controlled by RNA-binding proteins (RBPs) through interacting with their target RNAs in a sequence-dependent manner. In this study, we investigate the cytosolic functions of two neuronal RBPs, Ptbp2 and hnRNP R, which are essential for axon growth in motoneurons. Polypyrimidine tract binding protein 2 (Ptbp2) contributes to neuronal differentiation and axonogenesis by modulating different splicing programs to adjust the level of proteins involved in these processes. While the nuclear functions of Ptbp2 in alternative splicing have been studied in more detail, the cytosolic roles of Ptbp2 associated with axon growth have remained elusive. In the first part of the study, we show that Ptbp2 is present in cytosolic fractions of motoneurons including axons and axon terminals. Depletion of Ptbp2 impairs axon growth and growth cone maturation in cultured embryonic mouse motoneurons. Moreover, Ptbp2 knockdown affects the level of piccolo protein in the growth cone of cultured motoneurons. We detect Ptbp2 as a top interactor of the 3' UTR of the Hnrnpr transcript encoding the RBP hnRNP R. This interaction results in axonal localization of and thereby local translation of Hnrnpr mRNA in motoneurons. Consequently, axonal synthesis of hnRNP R was diminished upon depletion of Ptbp2 in motoneurons. We present evidence that Ptbp2 through cooperation with translation factor eIF5A2 controls hnRNP R synthesis. Additionally, we observe that re-expression of hnRNP R in Ptbp2-deficient motoneurons rescued axon growth defect while Ptbp2 overexpression failed to normalize the axon elongation defect observed in hnRNP R-deficient motoneurons. Our findings pinpoint axonal synthesized hnRNP R as a mediator of Ptbp2 functions in axon growth. In the second part of this study, we identify hnRNP R binds to the 3' UTR of microtubule-associated tau (Mapt) transcript encoding tau protein and regulates the axonal translocation and translation of Mapt mRNA. Tau protein has a central role in neuronal microtubule assembly and stability. However, in AD, the accumulation of abnormally hyperphosphorylated tau protein leads to axon outgrowth defects. Loss of hnRNP R reduces axonal tau protein but not the total level of tau. We observe that the brains of 5xFAD mice, as a mouse model of AD, deficient for hnRNP R contain lower phospho-tau and amyloid-β plaques. Likewise, Neurons treated with blocking antisense oligonucleotides (ASO) to prevent binding of hnRNP R to Mapt mRNA show reduced axonal Mapt mRNA and consequently newly synthesized tau protein levels. We show that blocking Mapt mRNA transport to axons impairs axon elongation. Our data thus suggest that reducing tau levels selectively in axons, a major subcellular site of tangle formation, might represent a novel therapeutic approach for the treatment of AD.