RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

BACKGROUND: TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. RESULTS: Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. CONCLUSIONS: Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

Silica nanoparticle uptake induces survival mechanism in A549 cells by the activation of autophagy but not apoptosis.

We report here an in vitro evaluation of silica nanoparticle uptake by lung epithelial cells (A549), the cytotoxic effect of the particles and we propose autophagy as possible survival strategy. The effect of surface charge, serum proteins and the influence of inhibitors on the uptake of 20 nm monodispersed nanoparticles with various functional groups are discussed. Uptake rate of the particles with various functional groups is demonstrated to be similar in the presence of serum proteins, while the uptake rate ranking is COOH>NH2>OH under serum free conditions. Our results suggest an actin-dependent, macropinocytotic uptake process that was also confirmed by scanning and transmission electron microscopy. In spite of the intensive active uptake, significant cytotoxic effect is detected only at relatively high concentrations (above 250 µg/mL). Blebbing of the cell surface is observed already at 5h of exposure and is shown to be related to autophagy rather than apoptotic cell death. The A549 cells display elevated levels of autophagosomes, however they do not express typical apoptosis markers such as increased amount of active caspase-3 and release of mitochondrial cytochrome C. Based on these results, we propose here an autophagic activity and cross-talk between autophagic and apoptotic pathways as a mechanism allowing the survival of A549 cells under exposure to silica nanoparticles.