hnRNP R regulates mitochondrial movement and membrane potential in axons of motoneurons.

Axonal mitochondria defects are early events in the pathogenesis of motoneuron disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. The RNA-binding protein hnRNP R interacts with different motoneuron disease-related proteins such as SMN and TDP-43 and has important roles in axons of motoneurons, including axonal mRNA transport. However, whether hnRNP R also modulates axonal mitochondria is currently unknown. Here, we show that axonal mitochondria exhibit altered function and motility in hnRNP R-deficient motoneurons. Motoneurons lacking hnRNP R show decreased anterograde and increased retrograde transport of mitochondria in axons. Furthermore, hnRNP R-deficiency leads to mitochondrial hyperpolarization, caused by decreased complex I and reversed complex V activity within the respiratory chain. Taken together, our data indicate a role for hnRNP R in regulating transport and maintaining functionality of axonal mitochondria in motoneurons.

Cytosolic Ptbp2 modulates axon growth in motoneurons through axonal localization and translation of Hnrnpr.

The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3' UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein.

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.

hnRNP R negatively regulates transcription by modulating the association of P-TEFb with 7SK and BRD4.

The P-TEFb complex promotes transcription elongation by releasing paused RNA polymerase II. P-TEFb itself is known to be inactivated through binding to the non-coding RNA 7SK but there is only limited information about mechanisms regulating their association. Here, we show that cells deficient in the RNA-binding protein hnRNP R, a known 7SK interactor, exhibit increased transcription due to phosphorylation of RNA polymerase II. Intriguingly, loss of hnRNP R promotes the release of P-TEFb from 7SK, accompanied by enhanced hnRNP A1 binding to 7SK. Additionally, we found that hnRNP R interacts with BRD4, and that hnRNP R depletion increases BRD4 binding to the P-TEFb component CDK9. Finally, CDK9 is stabilized upon loss of hnRNP R and its association with Cyclin K is enhanced. Together, our results indicate that hnRNP R negatively regulates transcription by modulating the activity and stability of the P-TEFb complex, exemplifying the multimodal regulation of P-TEFb by an RNA-binding protein.

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.

Keeping the balance: The noncoding RNA 7SK as a master regulator for neuron development and function.

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.

Interaction of 7SK with the Smn complex modulates snRNP production.

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.

Voltage-independent GluN2A-type NMDA receptor Ca2+ signaling promotes audiogenic seizures, attentional and cognitive deficits in mice.

The NMDA receptor-mediated Ca2+ signaling during simultaneous pre- and postsynaptic activity is critically involved in synaptic plasticity and thus has a key role in the nervous system. In GRIN2-variant patients alterations of this coincidence detection provoked complex clinical phenotypes, ranging from reduced muscle strength to epileptic seizures and intellectual disability. By using our gene-targeted mouse line (Grin2aN615S), we show that voltage-independent glutamate-gated signaling of GluN2A-containing NMDA receptors is associated with NMDAR-dependent audiogenic seizures due to hyperexcitable midbrain circuits. In contrast, the NMDAR antagonist MK-801-induced c-Fos expression is reduced in the hippocampus. Likewise, the synchronization of theta- and gamma oscillatory activity is lowered during exploration, demonstrating reduced hippocampal activity. This is associated with exploratory hyperactivity and aberrantly increased and dysregulated levels of attention that can interfere with associative learning, in particular when relevant cues and reward outcomes are disconnected in space and time. Together, our findings provide (i) experimental evidence that the inherent voltage-dependent Ca2+ signaling of NMDA receptors is essential for maintaining appropriate responses to sensory stimuli and (ii) a mechanistic explanation for the neurological manifestations seen in the NMDAR-related human disorders with GRIN2 variant-meidiated intellectual disability and focal epilepsy.

Absence of Plekhg5 Results in Myelin Infoldings Corresponding to an Impaired Schwann Cell Autophagy, and a Reduced T-Cell Infiltration Into Peripheral Nerves.

Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophages, which are modulated by T-lymphocytes. Here, we show that the NF-κB activator Pleckstrin homology containing family member 5 (Plekhg5) is involved in the regulation of both Schwann cell autophagy and recruitment of T-lymphocytes in peripheral nerves during motoneuron disease. Plekhg5-deficient mice show defective axon/Schwann cell units characterized by myelin infoldings in peripheral nerves. Even at late stages, Plekhg5-deficient mice do not show any signs of demyelination and inflammation. Using RNAseq, we identified a transcriptional signature for an impaired immune response in sciatic nerves, which manifested in a reduced number of CD4+ and CD8+ T-cells. These findings identify Plekhg5 as a promising target to impede myelin breakdown in demyelinating PNS disorders.

Author Correction: A systems view of spliceosomal assembly and branchpoints with iCLIP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of Tdp-43 disrupts the axonal transcriptome of motoneurons accompanied by impaired axonal translation and mitochondria function.

Protein inclusions containing the RNA-binding protein TDP-43 are a pathological hallmark of amyotrophic lateral sclerosis and other neurodegenerative disorders. The loss of TDP-43 function that is associated with these inclusions affects post-transcriptional processing of RNAs in multiple ways including pre-mRNA splicing, nucleocytoplasmic transport, modulation of mRNA stability and translation. In contrast, less is known about the role of TDP-43 in axonal RNA metabolism in motoneurons. Here we show that depletion of Tdp-43 in primary motoneurons affects axon growth. This defect is accompanied by subcellular transcriptome alterations in the axonal and somatodendritic compartment. The axonal localization of transcripts encoding components of the cytoskeleton, the translational machinery and transcripts involved in mitochondrial energy metabolism were particularly affected by loss of Tdp-43. Accordingly, we observed reduced protein synthesis and disturbed mitochondrial functions in axons of Tdp-43-depleted motoneurons. Treatment with nicotinamide rescued the axon growth defect associated with loss of Tdp-43. These results show that Tdp-43 depletion in motoneurons affects several pathways integral to axon health indicating that loss of TDP-43 function could thus make a major contribution to axonal pathomechanisms in ALS.

R-Roscovitine Improves Motoneuron Function in Mouse Models for Spinal Muscular Atrophy.

Neurotransmission defects and motoneuron degeneration are hallmarks of spinal muscular atrophy, a monogenetic disease caused by the deficiency of the SMN protein. In the present study, we show that systemic application of R-Roscovitine, a Cav2.1/Cav2.2 channel modifier and a cyclin-dependent kinase 5 (Cdk-5) inhibitor, significantly improved survival of SMA mice. In addition, R-Roscovitine increased Cav2.1 channel density and sizes of the motor endplates. In vitro, R-Roscovitine restored axon lengths and growth cone sizes of Smn-deficient motoneurons corresponding to enhanced spontaneous Ca2+ influx and elevated Cav2.2 channel cluster formations independent of its capability to inhibit Cdk-5. Acute application of R-Roscovitine at the neuromuscular junction significantly increased evoked neurotransmitter release, increased the frequency of spontaneous miniature potentials, and lowered the activation threshold of silent terminals. These data indicate that R-Roscovitine improves Ca2+ signaling and Ca2+ homeostasis in Smn-deficient motoneurons, which is generally crucial for motoneuron differentiation, maturation, and function.

A systems view of spliceosomal assembly and branchpoints with iCLIP.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites. We identified RNA binding proteins that crosslink to each peak, including known and candidate splicing factors. Moreover, we detected the use of over 40,000 BPs with strong sequence consensus and structural accessibility, which align well to nearby crosslinking peaks. We show how the position and strength of BPs affect the crosslinking patterns of spliceosomal factors, which bind more efficiently upstream of strong or proximally located BPs and downstream of weak or distally located BPs. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

Impaired Local Translation of ß-actin mRNA in Ighmbp2-Deficient Motoneurons: Implications for Spinal Muscular Atrophy with respiratory Distress (SMARD1).

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal motoneuron disorder in children with unknown etiology. The disease is caused by mutations in the IGHMBP2 gene, encoding a Super Family 1 (SF1)-type RNA/DNA helicase. IGHMBP2 is a cytosolic protein that binds to ribosomes and polysomes, suggesting a role in mRNA metabolism. Here we performed morphological and functional analyses of isolated immunoglobulin µ-binding protein 2 (Ighmbp2)-deficient motoneurons to address the question whether the SMARD1 phenotype results from de-regulation of protein biosynthesis. Ighmbp2-deficient motoneurons exhibited only moderate morphological aberrations such as a slight increase of axonal branches. Consistent with the rather mild phenotypic aberrations, RNA sequencing of Ighmbp2-deficient motoneurons revealed only minor transcriptome alterations compared to controls. Likewise, we did not detect any global changes in protein synthesis using pulsed SILAC (Stable Isotope Labeling by Amino acids in Cell culture), FUNCAT (FlUorescent Non-Canonical Amino acid Tagging) and SUnSET (SUrface SEnsing of Translation) approaches. However, we observed reduced ß-actin protein levels at the growth cone of Ighmbp2-deficient motoneurons which was accompanied by reduced level of IMP1/ZBP1, a known interactor of ß-actin mRNA. Fluorescence Recovery after Photobleaching (FRAP) studies revealed translational down-regulation of an eGFP-myr-ß-actin 3'UTR mRNA in growth cones. Local translational regulation of ß-actin mRNA was dependent on the 3' UTR but independent of direct Ighmbp2-binding to ß-actin mRNA. Taken together, our data indicate that Ighmbp2 deficiency results in local but modest disruption of protein biosynthesis which might partially contribute to the motoneuron defects seen in SMARD1.

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.

Optimized Whole Transcriptome Profiling of Motor Axons.

In highly polarized cells such as neurons, most RNA molecules are not randomly distributed but sorted into different compartments. So far, methods to analyze the transcriptome in distinct subcellular compartments are not well established. Here, we first describe the culturing of primary motoneurons in compartmentalized chambers to separate the axons from the somatodendritic compartment. Second, we introduce a method for whole transcriptome amplification followed by high-throughput sequencing to analyze the RNA composition of these two different compartments in neuronal cells.

Whole transcriptome profiling reveals the RNA content of motor axons.

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.

Recursive splicing in long vertebrate genes.

It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons.