TDP‐43, recently identified as a signature protein of the pathogenic inclusions in the brains cells of frontotemporal lobar degeneration patients, is a 43 kDa RNA‐binding protein. It has been known mainly as a nuclear factor capable of repressing transcription and promoting exon exclusion. TDP‐43 also forms distinct nuclear substructures linking different types of nuclear bodies. In this study, we provide the first evidence supporting TDP‐43 as a neuronal activity‐responsive factor in the dendrites of hippocampal neurons. In particular, TDP‐43 resides in the somatodendrites mainly in the form of RNA granules colocalized with the post‐synaptic protein PSD‐95. These granules also contain RNAs including at least the β‐actin mRNA and CaMKIIα mRNA. Furthermore, TDP‐43 is localized in the dendritic processing (P) body and it behaves as a translational repressor in an in vitro assay. Related to this, repetitive stimuli by KCl greatly enhance the colocalization of TDP‐43 granules with FMRP and Staufen 1, two RNA‐binding proteins known to regulate mRNA transport and local translation in neurons. These data together suggest that TDP‐43 is a neuronal activity‐responsive factor functioning in the regulation of neuronal plasticity, the impairment of which would lead to the development of certain forms of neurodegenerative diseases including frontotemporal lobar degeneration.

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.RNA metabolism is a complex process affecting the control of gene expression. In bacteria, a multicomponent ribonucleolytic complex termed the RNA degradosome (1-4) has been identified as playing an important role in the control of mRNA degradation (for recent reviews, see Refs. 5-11). The multicomponent complex consists of: the RNA endonuclease RNase E, whose activity is essential for Escherichia coli cell growth (12-14), RNA processing (15, 16), and degradation (17, 18); the 3Ј-5Ј exoribonuclease PNPase (19); RhlB RNA helicase (20); and enolase (21), an enzyme involved in the glycolytic pathway and other chaperonin proteins (3,22). Interestingly, in addition to mRNAs, highly structured, stable RNA fragments have also been found to be associated with RNA degradosome complexes (3, 23), which implies quality control by the RNA degradosome for the biogenesis of stable RNAs. Degradosome complexity and its cooperation with individual protein components acting on degradation-targeted RNA, in vivo, remains to be discovered.Various approaches revealing protein-protein interactions in the degradosome indicate that the C-terminal region of RNase E serves as a scaffold that directly binds PNPase, RhlB RNA helicase, and enolase (24,25). No other interactions among these component proteins have been detected (25) or reported. Recently, a mini-degradosome complex (26) containing the scaffold region (without the N-terminal enzymatic region of RNase E), RhlB RNA helicase, and PNPase was reconstituted in vitro. These experiments revealed a functional inte...

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[PSI] strains ͉ amyloid ͉ Sup35 A myloid is a generic class of ordered protein aggregates self-assembled by many unrelated proteins (1). X-ray diffraction studies reveal that amyloid fibers are rich in ␤-strands, which run perpendicular to the fiber axis, forming the characteristic cross-␤ pattern (2). One of the most intriguing features of the amyloid is structural polymorphism, whereby the same protein polypeptide can adopt distinct chain-folding patterns to give rise to a variety of cross-␤ structures (3). Structural plasticity of the amyloid underlies the prion strain phenomenon. Selfnucleating amyloid conformers faithfully maintain their distinct folds in the host but sometimes can adjust their structures when encountering different sequences (such as during interspecies transmission), thus giving rise to novel prion strains (4, 5). It is not well understood how distinct cross-␤ folding patterns are determined and what is the mechanism for their interconversion.The yeast prion [PSI] is a self-propagating amyloid aggregate of Sup35, the yeast translation-termination factor whose aggregation in the [PSI ϩ ] state results in enhanced read-through of nonsense mutations (6, 7). Structural polymorphism of Sup35 amyloids gives rise to [PSI] strains that exhibit distinctive nonsense-suppression efficiencies and differential compatibility with Sup35 mutations (8-11). With the ease of biochemical and genetic manipulation, [PSI] strains provide excellent experimental systems to study amyloid polymorphism. However, because in vitro assembled amyloid samples often contain multiple fiber morphologies, great care must be taken to ensure correct interpretation of experimental results. Attributing genetic observations to pure structural causes, however, is complicated by the interplay between amyloid structures and cellular machineries. Here, we design a synthesized approach to avoid these difficulties, combining biochemistry and yeast genetics to examine the amyloid structures of three [PSI] strains: [VH], [VK], and [VL] (11). By individually substituting residues 5-55 of Sup35 with proline and inserting glycine in front of every other residue in this segment, we identify strain-specific Sup35 sequences in vivo. The necessity of these sequences for encoding infectious prion structures is then investigated by infecting yeast with amyloid fibers of Sup35 fragments assembled in vitro. Our goal is to define the minimally required Sup35 sequences that form core structures of the three [PSI] strains to understand how the same polypeptides are recruited in different ways to assemble polymorphic amyloid structures. ResultsProline Substitutions. We employ proline substitutions to map possible ␤-structures along the polypeptide chain. Proline residues with the unique cyclic structure generate bulges in ␤-strands and destabilize periodic amyloid structures. A panel of 51 [psi Ϫ ] haploid mutants, each having a single proline substitution in the amino acid residues 5-55 of the SUP35 allele, was created by homologous recombination. ...