SMN regulates axonal local translation via miR-183/mTOR pathway.

Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn-mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.

Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits.

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of ß-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Phosphoproteomics.

Current analytical protein methods show phosphorylation to be the most ubiquitous, evolutionary conserved post-translational modification Post-Translational Modification (PTM). The reversible and transient nature of protein phosphorylation allows signal transduction pathways to carry out diverse cellular functions. From bacteria to humans, phosphorylation serves to modify protein function by altering protein stability, cellular location, substrate affinity, complex formation, and activity; thus allowing essential events such as cell cycle and growth to occur at precise times and locations. Phosphorylation controls a variety of events at many biological levels including: housekeeping activities controlled by single cells such as DNA transcription, cell-cycle regulation, and energy metabolism; and cellular processes that involve signaling between cells or the environment including such as neuronal migration and immune system recognition. This review summarizes state-of-the-art proteomics technologies available to study phosphorylation in biological systems. We highlight the tremendous steps the field has made in the last 5 years which allow quantitative global analyses while pointing out caveats in experimentation.

Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator.

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism.

Genetic and biochemical strategies for identifying Drosophila genes that function in circadian control.

Explicit biochemical models have been elaborated for the circadian oscillators of cyanobacterial, fungal, insect, and mammalian species. In contrast, much remains to be learned about how such circadian oscillators regulate rhythmic physiological processes. This article summarizes contemporary genetic and biochemical strategies that are useful for identifying gene products that have a role in circadian control.

A role for CK2 in the Drosophila circadian oscillator.

The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, we show that casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the beta subunit of CK2 and is predicted to perturb CK2beta subunit dimerization. It is associated with reduced beta subunit levels, indicative of a defect in alpha:beta association and production of the tetrameric alpha2:beta2 holoenzyme. Consistent with a direct action on the clock mechanism, we show that CK2beta is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator.