Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations.

FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates. In contrast, mutations in arginine display a severely altered conformation, static binding to RNA, and formation of large condensates, signifying the role of arginine in driving proper RNA interaction. Glycine mutations undergo rapid loss of fluidity, emphasizing the role of glycine in promoting fluidity. Strikingly, the nuclear import receptor Karyopherin-ß2 reverses the mutant defects and recovers the wild-type FUS behavior. We reveal two distinct mechanisms underpinning potentially disparate pathogenic pathways of ALS-linked FUS mutants.

Ubiquilin 2 modulates ALS/FTD-linked FUS-RNA complex dynamics and stress granule formation.

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.

Single-Molecule and Ensemble Methods to Probe Initial Stages of RNP Granule Assembly.

Ribonucleoprotein (RNP) granules are membraneless organelles, consisting of high local concentrations of RNA and proteins bearing intrinsically disordered regions (IDRs). They are formed by liquid-liquid phase separation (LLPS). In neurodegenerative diseases such as ALS, mutations in granule proteins such as FUS and TDP-43 accelerate abnormal liquid to solid transition of RNP granules, leading to formation of fiber-like structures. Methods to study granules must be carefully selected based on the stage of granule's life. Here we describe a strategic combination of single-molecule biophysical and ensemble biochemical techniques that may be employed to extract insightful information about early stages of RNP granule formation. Protein-RNA interaction and stoichiometry of the complex in the early soluble stage of RNP assembly can be probed by single-molecule FRET (smFRET) assay and electrophoretic mobility shift assay (EMSA), respectively. RNP-RNP interaction that likely contributes to RNP nucleation can be reported on by a smFRET-based RNA annealing assay. The next stage in the assembly pathway, that is, phase separation from diffused to liquid-like droplets, may be monitored by a phase separation assay. Finally, RNP granules isolated from mammalian cells can be investigated using a unique single-molecule pull-down (SiMPull) assay.

Dimerization of SLX4 contributes to functioning of the SLX4-nuclease complex.

The Fanconi anemia protein SLX4 assembles a genome and telomere maintenance toolkit, consisting of the nucleases SLX1, MUS81 and XPF. Although it is known that SLX4 acts as a scaffold for building this complex, the molecular basis underlying this function of SLX4 remains unclear. Here, we report that functioning of SLX4 is dependent on its dimerization via an oligomerization motif called the BTB domain. We solved the crystal structure of the SLX4BTB dimer, identifying key contacts (F681 and F708) that mediate dimerization. Disruption of BTB dimerization abrogates nuclear foci formation and telomeric localization of not only SLX4 but also of its associated nucleases. Furthermore, dimerization-deficient SLX4 mutants cause defective cellular response to DNA interstrand crosslinking agent and telomere maintenance, underscoring the contribution of BTB domain-mediated dimerization of SLX4 in genome and telomere maintenance.

Fanconi anemia proteins in telomere maintenance.

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.

SLX4 contributes to telomere preservation and regulated processing of telomeric joint molecule intermediates.

SLX4 assembles a toolkit of endonucleases SLX1, MUS81 and XPF, which is recruited to telomeres via direct interaction of SLX4 with TRF2. Telomeres present an inherent obstacle for DNA replication and repair due to their high propensity to form branched DNA intermediates. Here we provide novel insight into the mechanism and regulation of the SLX4 complex in telomere preservation. SLX4 associates with telomeres throughout the cell cycle, peaking in late S phase and under genotoxic stress. Disruption of SLX4's interaction with TRF2 or SLX1 and SLX1's nuclease activity independently causes telomere fragility, suggesting a requirement of the SLX4 complex for nucleolytic resolution of branched intermediates during telomere replication. Indeed, the SLX1-SLX4 complex processes a variety of telomeric joint molecules in vitro. The nucleolytic activity of SLX1-SLX4 is negatively regulated by telomeric DNA-binding proteins TRF1 and TRF2 and is suppressed by the RecQ helicase BLM in vitro. In vivo, in the presence of functional BLM, telomeric circle formation and telomere sister chromatid exchange, both arising out of nucleolytic processing of telomeric homologous recombination intermediates, are suppressed. We propose that the SLX4-toolkit is a telomere accessory complex that, in conjunction with other telomere maintenance proteins, ensures unhindered, but regulated telomere maintenance.

Defective repair of uracil causes telomere defects in mouse hematopoietic cells.

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.

SLX4 assembles a telomere maintenance toolkit by bridging multiple endonucleases with telomeres.

SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.

Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing.

The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNA(Leu). In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing.

Amino-acid-dependent shift in tRNA synthetase editing mechanisms.

Many aminoacyl-tRNA synthetases prevent mistranslation by relying upon proofreading activities at multiple stages of the aminoacylation reaction. In leucyl-tRNA synthetase (LeuRS), editing activities that precede or are subsequent to tRNA charging have been identified. Although both are operational, either the pre- or post-transfer editing activity can predominate. Yeast cytoplasmic LeuRS (ycLeuRS) misactivates structurally similar noncognate amino acids including isoleucine and methionine. We show that ycLeuRS has a robust post-transfer editing activity that efficiently clears tRNA(Leu) mischarged with isoleucine. In comparison, the enzyme's post-transfer hydrolytic activity against tRNA(Leu) mischarged with methionine is weak. Rather, methionyl-adenylate is cleared robustly via an enzyme-mediated pre-transfer editing activity. We hypothesize that, similar to E. coli LeuRS, ycLeuRS has coexisting functional pre- and post-transfer editing activities. In the case of ycLeuRS, a shift between the two editing pathways is triggered by the identity of the noncognate amino acid.

Characterization of benzoxaborole-based antifungal resistance mutations demonstrates that editing depends on electrostatic stabilization of the leucyl-tRNA synthetase editing cap.

The broad-spectrum benzoxaborole antifungal AN2690 blocks protein synthesis by inhibiting leucyl-tRNA synthetase (LeuRS) via a novel oxaborole tRNA trapping mechanism in the editing site. Herein, one set of resistance mutations is at Asp487 outside the LeuRS hydrolytic editing pocket, in a region of unknown function. It is located within a eukaryote/archaea specific insert I4, which forms part of a cap over a benzoxaborole-AMP that is bound in the LeuRS CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap of yeast LeuRS that is critical for tRNA deacylation. We hypothesize that this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis.

The microscopic origin of self-organized nanostripe pattern formation on an electropolished aluminium surface.

By correlating the experimental evidence obtained from atomic force microscopy, conventional x-ray diffraction, and a surface sensitive modified x-ray diffraction technique with the results of density functional theory based computations, we demonstrate that self-organized nanostripe patterns formed on the electropolished surface of aluminium originate as a consequence of relaxation and reconstruction of the new surfaces exposed and textural changes at the surface caused by the dissolution during polishing.

La estrategia de los niños sanos: experiencias de la implementación de AIEPI comunitario en Bolivia / Es

Este libro narra la experiencia pionera del PLan Internacional en la implementacion del componente comunitario de la estrategia AIEPI para la reduccion de la mortalidad infantil en menores de cinco años en Bolivia...

La estrategia de los niños sanos: experiencias de la implementación de AIEPI comunitario en Bolivia / Es

Este libro narra la experiencia pionera del PLan Internacional en la implementacion del componente comunitario de la estrategia AIEPI para la reduccion de la mortalidad infantil en menores de cinco años en Bolivia...