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Tools to understand how the spliceosome functions in vivo have lagged behind advances in the structural biology of the spliceosome. Here, methods are described to globally profile spliceosome-bound pre-mRNA, intermediates, and spliced mRNA at nucleotide resolution. These tools are applied to three yeast species that span 600 million years of evolution. The sensitivity of the approach enables the detection...
Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination...
Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically...
Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in...
We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption...
During each life cycle, germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here, we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis...
Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX 3 CR1 CreER mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function...
Parkinson’s disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and...
Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat...
Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target...
Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting...
Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5′ flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report...
The centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from...
Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis...
Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence...
Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds...
Proper connections between centromeres and spindle microtubules are of critical importance in ensuring accurate segregation of the genome during cell division. Using an in vitro approach based on the sequence-specific budding yeast centromere, we identified a complex of the chromosomal passenger proteins Bir1 and Sli15 (Survivin and INCENP) that links centromeres to microtubules. This linkage does...
The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic...
Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes—i.e., protein acyltransferases (PATs)—were identified...
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