Loss of full-length hnRNP R isoform impairs DNA damage response in motoneurons by inhibiting Yb1 recruitment to chromatin.

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 (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/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 γ-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.

hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons.

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.

Metabolic plasticity in a Pde6bSTOP/STOP retinitis pigmentosa mouse model following rescue.

Retinitis pigmentosa (RP) is a hereditary retinal disease characterized by progressive photoreceptor degeneration, leading to vision loss. The best hope for a cure for RP lies in gene therapy. However, given that RP patients are most often diagnosed in the midst of ongoing photoreceptor degeneration, it is important to determine how the retinal proteome changes as RP disease progresses, and to identify which changes can be prevented, halted, or reversed by gene therapy. Here, we used our Pde6b-deficient RP gene therapy mouse model and demonstrated that Pde6b gene restoration led to a novel form of homeostatic plasticity in rod phototransduction which functionally compensates for the decreased number of rods. By profiling protein levels of metabolic genes and measuring metabolites, we observed an upregulation of proteins associated with oxidative phosphorylation in mutant and treated photoreceptors. Thus, the metabolic demands of the retina differ in our Pde6b-deficient RP mouse model and are not rescued by gene therapy treatment. These findings provide novel insights into features of both RP disease progression and long-term rescue with gene therapy.

Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy.

Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 "cluster-like" formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.