Pumilio2 and Staufen2 selectively balance the synaptic proteome.

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission.

Isolation and Characterization of Endogenous RNPs from Brain Tissues.

Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprotein particles that then can be analyzed by mass spectrometry (for proteins composition) and microarray or RNA sequencing technologies (for target mRNAs).

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

Immunocytochemical studies on the effect of 405-nm low-power laser irradiation on human-derived A-172 glioblastoma cells.

The application of low-power laser irradiation (LLI) affects the cell cycle and cell proliferation in various kinds of cells. LLI at a wavelength of 808 nm and a power of 30 mW has been found to significantly decrease the proliferation rate of cells of the human-derived glioblastoma cell line A-172. To determine if this effect of LLI is specific to 808-nm LLI, the present study was designed to reveal the effects of 405-nm LLI under the same experimental conditions. A-172 glioblastoma cells were cultured in 96-well plates according to the conventional protocol. Two different schedules of 405-nm LLI (27 mW) were tested: longer periods of 20, 40 and 60 min and shorter periods of 1, 2, 3, 5, 10 and 15 min. Cells on a digital image displayed on a computer monitor were counted and the proliferation ratio was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) staining. Annexin-V-FLUOS staining and acridine-orange/ethidium-bromide staining were in an immunocytochemical assay to determine if cells were viable or dead (due to apoptosis or necrosis). Cell counting and MTT staining showed that longer 405-nm LLI significantly suppressed the proliferation of A-172 cells at 48 h after LLI (p < 0.05 or p < 0.01) and that the effect of LLI tended to be dose-dependent with morphological changes including cell death. At 90 min after LLI, shorter 405-nm LLI caused necrotic as well as apoptotic cell death, and these effects depended on irradiation time, power and energy density. Detailed analysis revealed that this lethal effect occurred after LLI and was not sustainable. It is concluded that 405-nm LLI has a lethal effect on human-derived glioblastoma A-172 cells, that is different from the suppressive effect without morphological changes induced by 808-nm LLI.