Regulated dicing of pre-mir-144 via reshaping of its terminal loop.

Although the route to generate microRNAs (miRNAs) is often depicted as a linear series of sequential and constitutive cleavages, we now appreciate multiple alternative pathways as well as diverse strategies to modulate their processing and function. Here, we identify an unusually profound regulatory role of conserved loop sequences in vertebrate pre-mir-144, which are essential for its cleavage by the Dicer RNase III enzyme in human and zebrafish models. Our data indicate that pre-mir-144 dicing is positively regulated via its terminal loop, and involves the ILF3 complex (NF90 and its partner NF45/ILF2). We provide further evidence that this regulatory switch involves reshaping of the pre-mir-144 apical loop into a structure that is appropriate for Dicer cleavage. In light of our recent findings that mir-144 promotes the nuclear biogenesis of its neighbor mir-451, these data extend the complex hierarchy of nuclear and cytoplasmic regulatory events that can control the maturation of clustered miRNAs

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins

TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach <i>via</i> atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation

Disassembly of YB-1 amyloid-like fibrils at physiological ionic strength.

<p>(A) and (B), YB-1 (56.8 µM) was incubated with 0.15 M or 2 M KCl for 24 h. An aliquot of YB-1 pre-incubated with 2 M KCl was diluted to 0.15 M KCl. Remaining samples were diluted to the same final protein concentration (4.26 µM) with appropriate KCl solutions to keep the salt concentration unchanged. The samples were incubated for 1 h at room temperature and analyzed by (A), ThT fluorescence (means and SD are shown (n = 3), and *** indicates <i>t</i>-test <i>p</i><0.001) and (B), EM imaging. (C), YB-1 (30 µM) was incubated with 0.15 M KCl or 2 M LiCl for 24 h. An aliquot of YB-1 pre-incubated with 2 M LiCl was dialyzed against 0.15 M KCl. Fibril formation was visualized by AFM imaging. Scale bars are 0.4 µm. Ionic strength conditions during the incubation are indicated.</p

Fibril formation by YB-1 and its fragments under physiological conditions.

<p>EM images of YB-1 (A), YB-1_1–219 (B), YB-1_1–129 (C), and YB-1_52–129 (D) (10 µM) incubated in the presence of 0.15 M KCl for 92 h. Scale bars are 0.4 µm.</p

Electrophoretic analysis of YB-1 and its fragments before and after incubation.

<p>YB-1 and its fragments before (−) and after (+) 24 h incubation in 2 M LiCl were subjected to 13% SDS-PAGE in a tris-tricine buffer system and stained with Coomassie brilliant blue. Protein molecular weight markers are shown (lane M).</p

The domain organization of YB-1.

<p>(A), Prediction of structured and intrinsically disordered regions of YB-1 by IsUnstruct algorithm. Disorder is assigned to score values greater than or equal to 0.5. CSD is highlighted by gray shading. (B), The tertiary structure of CSD and sketched terminal domains. (C), YB-1 and its fragments used in the study. The indicated regions belong to different YB-1 domains.</p

YB-1 forms amyloid-like fibrils.

<p>(A), Congo red staining of YB-1 fibrils. YB-1 (∼1.4 mM) was incubated under high ionic strength conditions (2 M LiCl) for 72 h. Photographs were taken in a bright-field mode (left) or under cross-polarized light (right). (B), Circular dichroism spectroscopy of YB-1. Spectra were obtained for 30 µM YB-1 in 0.15 M KCl (solid line) and after incubation with 1 M MgSO₄ (dotted line). (C), X-ray diffraction of an oriented YB-1 fibril sample.</p