Progressive Motor and Non-Motor Symptoms in Park7 Knockout Zebrafish.

DJ-1 is a redox sensitive protein with a wide range of functions related to oxidative stress protection. Mutations in the park7 gene, which codes for DJ-1 are associated with early onset familial Parkinson's disease and increased astrocytic DJ-1 levels are found in pathologic tissues from idiopathic Parkinson's disease. We have previously established a DJ-1 knockout zebrafish line that developed normally, but with aging the DJ-1 null fish had a lowered level of tyrosine hydroxylase, respiratory mitochondrial failure and a lower body mass. Here we have examined the DJ-1 knockout from the early adult stage and show that loss of DJ-1 results in a progressive, age-dependent increase in both motoric and non-motoric symptoms associated to Parkinson's disease. These changes coincide with changes in mitochondrial and mitochondrial associated proteins. Recent studies have suggested that a decline in NAD+ can contribute to Parkinson's disease and that supplementation of NAD+ precursors may delay disease progression. We found that the brain NAD+/NADH ratio decreased in aging zebrafish but did not correlate with DJ-1 induced altered behavior. Differences were first observed at the late adult stage in which NAD+ and NADPH levels were decreased in DJ-1 knockouts. Considering the experimental power of zebrafish and the development of Parkinson's disease-related symptoms in the DJ-1 null fish, this model can serve as a useful tool both to understand the progression of the disease and the effect of suggested treatments.

Detecting translational regulation by change point analysis of ribosome profiling data sets.

Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While during normal translation, ribosome density is constant along the length of the mRNA coding region, this can be altered in response to translational regulatory events. In the present study, we developed a method to detect translational regulation of individual mRNAs from their ribosome profiles, utilizing changes in ribosome density. We used mathematical modeling to show that changes in ribosome density should occur along the mRNA at the point of regulation. We analyzed a Ribo-Seq data set obtained for mouse embryonic stem cells and showed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introduced by deep-sequencing and alignment artifacts. After normalization, we applied a change point algorithm to detect changes in ribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained from the UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. In particular, we detected several mRNAs with known post-transcriptional regulation, e.g., premature termination for selenoprotein mRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful for identification of new transcript isoforms.

A bending rigidity parameter for stress granule condensates.

Interfacial tension plays an important role in governing the dynamics of droplet coalescence and determining how condensates interact with and deform lipid membranes and biological filaments. We demonstrate that an interfacial tension-only model is inadequate for describing stress granules in live cells. Harnessing a high-throughput flicker spectroscopy pipeline to analyze the shape fluctuations of tens of thousands of stress granules, we find that the measured fluctuation spectra require an additional contribution, which we attribute to elastic bending deformation. We also show that stress granules have an irregular, nonspherical base shape. These results suggest that stress granules are viscoelastic droplets with a structured interface, rather than simple Newtonian liquids. Furthermore, we observe that the measured interfacial tensions and bending rigidities span a range of several orders of magnitude. Hence, different types of stress granules (and more generally, other biomolecular condensates) can only be differentiated via large-scale surveys.

Peptide-Based Coacervate-Core Vesicles with Semipermeable Membranes.

Coacervates droplets have long been considered as potential protocells to mimic living cells. However, these droplets lack a membrane and are prone to coalescence, limiting their ability to survive, interact, and organize into higher-order assemblies. This work shows that tyrosine-rich peptide conjugates can undergo liquid-liquid phase separation in a well-defined pH window and transform into stable membrane-enclosed protocells by enzymatic oxidation and cross-linking at the liquid-liquid interface. The oxidation of the tyrosine-rich peptides into dityrosine creates a semipermeable, flexible membrane around the coacervates with tunable thickness, which displays strong intrinsic fluorescence, and stabilizes the coacervate protocells against coalescence. The membranes have an effective molecular weight cut-off of 2.5 kDa, as determined from the partitioning of small dyes and labeled peptides, RNA, and polymers into the membrane-enclosed coacervate protocells. Flicker spectroscopy reveals a membrane bending rigidity of only 0.1kB T, which is substantially lower than phospholipid bilayers despite a larger membrane thickness. Finally, it is shown that enzymes can be stably encapsulated inside the protocells and be supplied with substrates from outside, which opens the way for these membrane-bound compartments to be used as molecularly crowded artificial cells capable of communication or as a vehicle for drug delivery.

Deep conservation of ribosome stall sites across RNA processing genes.

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

Unrestrained ESCRT-III drives micronuclear catastrophe and chromosome fragmentation.

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.

Identification of novel androgen-regulated pathways and mRNA isoforms through genome-wide exon-specific profiling of the LNCaP transcriptome.

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.

Alternative RNA splicing regulation in the testis.

Alternative splicing regulation has been shown to be critically important for several developmental pathways. It is particularly prevalent in the testis, which is the site of an extensive adult developmental programme. Alternative splicing is controlled by a splicing code, in which transcripts respond to subtle cell type-specific variations in positive and negative trans-acting RNA-binding proteins according to their unique set of binding sites for these proteins. Because of their unique combinations of cis-acting sequence elements, specific transcripts are able to respond individually to this code. In this review, we discuss how this code may be deciphered in germ cells to mediate a splicing response.