Project description:In neurons, mRNAs and associated RNA-binding proteins assemble into ribonucleoprotein (RNP) granules essential to regulate mRNA trafficking, local translation, and turnover. Dysregulation of RNA-protein condensation can disturb synaptic plasticity. We report that the novel lncRNA mimi is a constitutive and essential component of large cytoplasmic condensates (RNP granules) in fly neurons that are biochemically enriched by differential centrifugation. Here, employing relative iBAQ quantification we carry out a differential proteomic analysis of cytoplasmic RNP granules in wild-type versus delta-mimi mutant fly brains. Brain lysates from wild-type and mutant flies serve as a general proteome input control.
Project description:Ribonucleoprotein (RNP) granules are non-membrane bound organelles thought to assemble by protein-protein interactions of RNA-binding proteins. We present evidence that a wide variety of RNAs self-assemble in vitro into either liquid-liquid phase separations or more stable assembles, referred to as RNA tangles. Self-assembly in vitro is affected by RNA length, ionic conditions, and sequence. Remarkably, self-assembly of yeast RNA in vitro under physiological salt mimics the composition of mRNAs within yeast stress granules. This suggests that the biophysical principles that drive RNA self-assembly in vitro contribute to determining the stress granule transcriptome. Consistent with RNA self-assembly contributing to RNP granule formation, an excess of purified RNA injected into C. elegans syncytium coalesces into droplet-like assemblies. We suggest that diverse RNA-RNA interactions in trans contribute to RNP granule formation and may explain the prevalence of large RNA-protein assemblies in eukaryotic cells.
Project description:Maternal RNA degradation is critical for embryogenesis and is tightly controlled by maternal RNA-binding proteins. Fragile X mental-retardation protein (FMR1) binds target mRNAs to form ribonucleoprotein (RNP) complexes/granules that control various biological processes, including early embryogenesis. However, how FMR1 recognizes target mRNAs and how FMR1-RNP granule assembly/disassembly regulates FMR1-associated mRNAs remain elusive. Here we show that Drosophila FMR1 preferentially binds mRNAs containing m6A-marked “AGACU” motif with high affinity to contributes to maternal RNA degradation. The high-affinity binding largely depends on a hydrophobic network within FMR1 KH2 domain. Importantly, this binding greatly induces FMR1 granule condensation to efficiently recruit unmodified mRNAs. The degradation of maternal mRNAs then causes granule de-condensation, allowing normal embryogenesis. Our findings reveal that sequence-specific mRNAs instruct FMR1-RNP granules to undergo a dynamic phase-switch, thus contributes to maternal mRNA decay. This mechanism may represent a general principle that regulated RNP-granules control RNA processing and normal development.
Project description:P-bodies (PB) are cytoplasmic RNP complexes that aggregate into foci when cells are exposed to stress. While the core components and stress response of PB and related RNP granules are conserved, it remains unclear how and why cells assemble mRNP complexes into granule foci during stress. We use mass spectrometry and antibody-based microarray to analyze proteins and RNA, respectively, that are immunoisolated with the core PB protein Dhh1-GFP. Analysis of the RNA associated with Dhh1-GFP immunoisolate reveals an enrichment of mitochondrial catalytic RNPs complex, suggesting a role for PB in mitochondrial RNA processing.
Project description:T bruce were heat shocked for 1 h at 41C then granules were prepared as per Fritz et al. 2015. After spinning down cytoskeletons with trapped contents and lysing them we sequenced the supernatant (trapped RNA) and pellet (granule) fractions. Input total RNA is included for comparison.
Project description:Processing of primary transcripts in trypanosomes requires trans splicing and polyadenylation, and at least for the poly(A) polymerase gene, also internal cis splicing. The trypanosome U1 snRNA, which is most likely a cis-splicing specific component, is unusually short and has a relatively simple secondary structure. Here, we report the identification of three specific protein components of the Trypanosoma brucei U1 snRNP, based on mass spectrometry and confirmed by in vivo epitope tagging and in vitro RNA binding. Both T.brucei U1-70K and U1C are only distantly related to known counterparts from other eukaryotes. The T.brucei U1-70K protein represents a minimal version of 70K, recognizing the first loop sequence of U1 snRNA with the same specificity as the mammalian protein. The trypanosome U1C-like protein interacts with 70K directly and binds the 5' terminal sequence of U1 snRNA. Surprisingly, instead of U1A we have identified a novel U1 snRNP-specific protein, TbU1-24K. U1-24K lacks a known RNA-binding motif and integrates in the U1 snRNP via interaction with U1-70K. These data result in a model of the trypanosome U1 snRNP, which deviates substantially from our classical view of the U1 particle and may reflect the special requirements for splicing of a small set of cis-introns in trypanosomes.
Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate SLBP RNP from polyribosomal fractions of HeLa S3 lysates. Mock immunoprecipitation were also performed and serve as a negative control.
Project description:UV cross-linking and immunoprecipiatation with high throughput sequencing of cytoplasmic SLBP RNP Polyclonal α-SLBP antibody was used to immunoprecipitate UV-crosslinked SLBP RNP from cytoplasmic HeLa lysates. Mock immunoprecipitation were also performed and serve as a negative control.
Project description:P-bodies (PB) are cytoplasmic RNP complexes that aggregate into foci when cells are exposed to stress. While the core components and stress response of PB and related RNP granules are conserved, it remains unclear how and why cells assemble mRNP complexes into granule foci during stress. We use mass spectrometry and antibody-based microarray to analyze proteins and RNA, respectively, that are immunoisolated with the core PB protein Dhh1-GFP. Analysis of the RNA associated with Dhh1-GFP immunoisolate reveals an enrichment of mitochondrial catalytic RNPs complex, suggesting a role for PB in mitochondrial RNA processing. RNA from 7 anti-GFP immunoisolations as well as total (input) RNA for each experiment was prepared. The seven samples include one mock IP from a strain containing GFP alone as well as 6 from Dhh1-GFP strains, two replicates from a (+) glucose condition and four replicates from a (-) glucose condition in which Dhh1-GFP forms cytoplasmic foci. 5ug of total RNA and 200ng IP RNA was hybridized to cutsom Agilent microarrays and detected using the S9.6 monoclonal antibody to RNA:DNA hybrids (ATCC clone ) and a Cy3 labeled anti-mouse secondary antibody.