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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Nup170 with/without Brl1 degradation (DIA)

ABSTRACT: This project was aimed at investigating nuclear pore complex biogenesis upon inducible degradation of Brl1 using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, the nuclear pore complex was purified through endogenously tagged Nup170 at several time points following auxin inducible degradation of Brl1. Simultaneously with the degradation of Brl1, newly synthesized proteins were metabolically labeled using a SILAC approach. The metabolic labeling of all nuclear pore complex constituents was assessed. Proteins that depend on Brl1 for their assembly show delayed metabolic labeling.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Federico Uliana

LAB HEAD: Federico Uliana

PROVIDER: PXD032024 | Pride | 2022-08-17

REPOSITORIES: pride

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			Action	DRS
	211111_QE1_ulianaf_KW_05_01.raw	Raw
	211111_QE1_ulianaf_KW_05_02.raw	Raw
	211111_QE1_ulianaf_KW_05_03.raw	Raw
	211111_QE1_ulianaf_KW_05_05.raw	Raw
	211111_QE1_ulianaf_KW_05_06.raw	Raw

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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Brl1 (DIA)

Project description:This project was aimed at analyzing the binding of Brl1 to the nuclear pore complex during the assembly process using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, we affinity purified Brl1 at several time points following SILAC metabolic labeling in growing S. cerevisiae cultures. Then, we analyzed the metabolic labeling for all co-purified nucleoporins and used these labeling kinetics to infer the timing of Brl1 binding during the native nuclear pore complex assembly.

2022-08-17 | PXD032017 | Pride

An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - Lysate intermixing text (DIA)

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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae – targeted analysis of cell lysate (PRM)

Project description:The project was aimed at assessing the metabolic labeling of NUPs in the full lysate upon SILAC labeling of growing yeast cultures, 90 min post labeling. This experiment was performed to exclude that the systematic labeling differences that we observed in KARMA assays with Brl1 bait were present already in the source lysate.

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The histone acetyltransferase Mst2 prevents epigenetic silencing via specific acetylation of the ubiquitin ligase Brl1 [microarray data]

Project description:Faithful propagation of distinct chromatin states is crucial to maintain cellular identity. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report the discovery of a self-reinforcing feedback loop that preserves the transcription-competent state of RNA polymerase II transcribed genes. We found that the chromatin reader Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3 marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides histone H3K14, Mst2 specifically acetylates Brl1, a component of the histone H2B ubiquitin ligase complex (HULC). Brl1 acetylation results in increased histone H2B ubiquitination, which together with H3K14ac positively feeds back on transcription and prevents assembly of ectopic heterochromatin. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

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The histone acetyltransferase Mst2 protects active chromatin from epigenetic silencing by acetylating the ubiquitin ligase Brl1

Project description:Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we discover a self-reinforcing feedback loop that preserves the transcription-competent state of RNA polymerase II transcribed genes. We found that the chromatin reader Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3 marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation results in increased histone H2B ubiquitination, which together with H3K14ac positively feeds back on transcription and prevents assembly of ectopic heterochromatin. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

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The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome [RNA-Seq]

Project description:Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome, however, the way they are recognized and targeted to the exosome is not fully understood. The recently identified Schizosaccharomyces pombe MTREC complex has been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs. Here, we report that the MTREC complex is also the major nuclear exosome targeting complex for CUTs and unspliced mRNAs. MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and -processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of aberrant, cryptic transcripts. RNA sequencing of WT and mutant S.pombe strains, processed data is normalized to median non-intron containing gene-expression, no replicates

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2023-04-01 | GSE200715 | GEO

The WNT-dependent gating of MYC requires non-canonical functions of a distal CTCF binding site

Project description:Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer (OSE)-mediated gating of MYC to the nuclear pore. This process enables MYC transcripts to escape the higher degradation rate in the nucleus compared to the cytoplasm. By targeted mutation, we show here that a single OSE-specific CTCF binding site (CTCFBS) mediates the WNT-regulated increased nuclear export rate of mRNAs derived from its interacting MYC and FAM49B loci, located >0.5 and >2.6 mbp distal of the OSE. The CTCFBS coordinated OSE-MYC proximity with their peripheral localisation, and mediated the WNT-dependent recruitment of the nuclear pore anchor AHCTF1 to the OSE. Furthermore, the CTCFBS up-regulated CCAT1 eRNA transcription, implicated in OSE-MYC interactions, correlating with an enhanced ability of the OSE to reach the nuclear periphery. We submit that WNT-dependent gating of MYC requires CTCF complexed with the OSE to coordinate multiple steps of the gating process.

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