The Ccr4-Not complex has been implicated in the control of multiple steps of mRNA metabolism; however, its functions in transcription remain ambiguous. The discovery that Ccr4/Pop2 is the major cytoplasmic mRNA deadenylase and the detection of Not proteins within mRNA processing bodies have raised questions about the roles of the Ccr4-Not complex in transcription. Here we firmly establish Ccr4-Not as a positive elongation factor for RNA polymerase II (RNAPII). The Ccr4-Not complex is targeted to the coding region of genes in a transcription-dependent manner similar to RNAPII and promotes elongation in vivo. Furthermore, Ccr4-Not interacts directly with elongating RNAPII complexes and stimulates transcription elongation of arrested polymerase in vitro. Ccr4-Not can reactivate backtracked RNAPII using a mechanism different from that of the well-characterized elongation factor TFIIS. While not essential for its interaction with elongation complexes, Ccr4-Not interacts with the emerging transcript and promotes elongation in a manner dependent on transcript length, although this interaction is not required for it to bind RNAPII. Our comprehensive analysis shows that Ccr4-Not directly regulates transcription, and suggests it does so by promoting the resumption of elongation of arrested RNAPII when it encounters transcriptional blocks in vivo.
The 12 subunit Swi/Snf chromatin remodeling complex is conserved from yeast to humans. It functions to alter nucleosome positions by either sliding nucleosomes on DNA or evicting histones. Interestingly, 20% of all human cancers carry mutations in subunits of the Swi/Snf complex. Many of these mutations cause protein instability and loss, resulting in partial Swi/Snf complexes. Though several studies have shown that histone acetylation and activator dependent recruitment of Swi/Snf regulate its function, it is less well understood how subunits regulate stability and function of the complex. Using functional proteomic and genomic approaches we have assembled the network architecture of yeast Swi/Snf. In addition, we find that subunits of the Swi/Snf complex regulate occupancy of the catalytic subunit Snf2, thereby modulating gene transcription. Our findings have direct bearing on how cancer-causing mutations in orthologous sub-units of human Swi/Snf may lead to aberrant regulation of gene expression by this complex.
Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N-and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.The regulation of gene expression in eukaryotes is a complex series of events that is well coordinated by different cellular machineries. Central to this process is the regulation of mRNA metabolism. It begins with transcription followed by pre-mRNA processing in the nucleus, nucleo-cytoplasmic transport, translation, and finally, the controlled degradation of mRNA.mRNA degradation is one of the important means of mRNA quality control. In eukaryotic cells two major pathways have evolved for mRNA degradation; they are non-sense-mediated decay, which is deadenylation-independent and the more ubiquitously used deadenylation-dependent pathway (1). Nonsense-mediated decay is the process of choice for quick and efficient removal of transcripts with non-sense codons, unspliced introns, or extended 3Ј-UTR (2). The deadenylationdependent pathway begins with the removal of the 3Ј-poly(A) tail by 3Ј-5Ј exonucleases Ccr4 and Pop2 or poly(A) nuclease (3-7). This is followed by removal of the 5Ј-m 7 -guanosine triphosphate cap by the Dcp2/Dcp1 decapping complex (8, 9). The binding of Dcp1/Dcp2 to the 5Ј cap and decapping are enhanced by a complex of seven Lsm (like-Sm) proteins (Lsm1 through Lsm7), Pat1, Edc3,. The final step in the pathway involves degradation of the decapped and deadenylated RNA by the 5Ј-3Ј exonuclease Xrn1 and by the exosome that acts in the 3Ј-5Ј direction (16,17). Proteins involved in mRNA turnover are localized to distinct foci in the cytoplasm call...
Modifications of histones play important roles in balancing transcriptional output. The discovery of acyl marks, besides histone acetylation, has added to the functional diversity of histone modifications. Since all modifications use metabolic intermediates as substrates for chromatin modifying enzymes, the prevalent landscape of histone modifications in any cell type is a snapshot of its metabolic status. Here we review some of the current findings of how differential use of histone acylations regulates gene expression as response to metabolic changes and differentiation programs.
KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification.
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