Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component.

Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS).

Analysis of developmental gene expression using smFISH and staging of embryos.

Regulation of transcription during embryogenesis is key to development and differentiation. To study transcript expression throughout embryogenesis at single-molecule resolution, we developed a high-throughput single-molecule fluorescence in situ hybridization (smFISH) method that relies on computational methods to developmentally stage embryos and quantify individual mRNA molecules in single embryos. We applied our system to , a zygotically transcribed gene essential for hermaphrodite development and dosage compensation.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing.

A Multiplexed SEC-MS Approach to Systematically Study the Interplay Between Protein Assembly-States and Phosphorylation Events.

Protein molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly-states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly-states.

Condensin DC loads and spreads from recruitment sites to create loop-anchored TADs in .

Condensins are molecular motors that compact DNA via linear translocation. In , the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of ecruitment lements on the -chromosome () and is required for the formation of topologically associating domains (TADs).

The histone H4 lysine 20 demethylase DPY-21 regulates the dynamics of condensin DC binding.

Condensin is a multi-subunit structural maintenance of chromosomes (SMC) complex that binds to and compacts chromosomes. Here, we addressed the regulation of condensin binding dynamics using Caenorhabditis elegans condensin DC, which represses X chromosomes in hermaphrodites for dosage compensation. We established fluorescence recovery after photobleaching (FRAP) using the SMC4 homolog DPY-27 and showed that a well-characterized ATPase mutation abolishes DPY-27 binding to X chromosomes.

Binding of an -Specific Condensin Correlates with a Reduction in Active Histone Modifications at Gene Regulatory Elements.

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In , a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing.

Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation.

In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in . We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific.