Functional analysis of the CAF1 protein

The CAF1 protein is a component of the CCR4-NOT deadenylase complex. While yeast CAF1 displays deadenylase activity, this activity is not required for its function in vivo, and CCR4 is the primary deadenylase in the complex. In order to identify CAF1-specific functional regions required for deadenylation in vivo, we targeted for mutagenesis six regions of CAF1 that are specifically conserved among CAF1 orthologs. Defects in three regions of the CAF1 protein (residues 173-175, residues 255-257 and residues 340-342, alleles caf1-1, caf1-3 and caf1-6, respectively) were found to dramatically reduce the rate of deadenylation in vivo and to result in typical caf1 deletion phenotypes without critically affecting the ability of CAF1 to bind CCR4. In contrast, defects in residues 213-215, which were defined as the site required for binding CCR4 (caf1-2 allele), reduced the rate of deadenylation to a lesser extent than did caf1-1, -3 or -6 and its in vivo phenotypes were correspondingly less severe than these other alleles. The caf1-1, -3, and -6 alleles or a caf1 deletion, unlike that of ccr4 or caf1-2, were synthetically lethal with defects in DHH1, a decapping regulator that is involved in controlling translation, and with certain defects in poly(A) binding protein (PAB1) that display decreased rates of translation and deadenylation. A caf1 deletion also had a more significant effect on translation than did ccr4. These and other genetic experiments suggest a role for CAF1 in translation separate from that of CCR4. Conversely, a pab1 translation defect that did not affect deadenylation by itself or with ccr4 severely blocked deadenylation when coupled with a caf1 deletion. These results support a role for CAF1 in communications between translation and deadenylation that are required for both processes

eIF5A Promotes Translation Elongation, Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions

Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle

Splicing factor SRSF3 represses translation of p21(cip1/waf1) mRNA

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3′UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3′UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3. © 2022, The Author(s).TRU

MeCP2 regulates gene expression through recognition of H3K27me3.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation

Effects of individual Cullin silencing in genome integrity.

<p><b>A</b>. γ-H2AX foci induction was measured in HEY cells treated siRNAs against indicated cullins. Knockdown efficiency is shown in right. <b>B</b>. Formation of double strand breaks were measured using neutral comet assay, in HEY cells treated with siRNAs against indicated cullins.</p

Inhibiting CUL2 neddylation leads to impaired G1-S transition.

<p><b>A</b>. Double thymidine block experiments were performed in HEY cells treated with DMSO control or MLN4924 and UBE2M siRNA. See Experimental procedure for detailed protocol. The red line was established by selecting the peak value of the cells in G1 (2N) for the control siRNA sample at the zero hour time point. The red line was then kept constant between samples to provide a means of comparison. <b>B</b>. Double thymidine block experiments were performed in HEY cells individually knockdown with indicated cullins. <b>C</b>. Expression of siRNA-resistant CUL2 wild type (WT), but not the empty vector (EV) nor the CUL2 mutant (C689R; ΔNedd8 in the figure), partially rescues the G1-S arrest phenotype. CUL2 siRNA #3 targets the 3′UTR of the CUL2 mRNA. Western blot confirms the knockdown efficiency and ectopic expression of CUL2 proteins. <b>D</b>. Double thymidine block experiments were performed using the HCT116 wild type or p21-/- cells that are treated with either control or CUL2 siRNAs. <b>E</b>. Induction rate of RAD51 foci was measured in HEY cells treated with control or CUL2 siRNAs. The counting was <i>normalized</i> to the 0 time point to indicate the fold increase.</p

MeCP2 regulates gene expression through recognition of H3K27me3.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation

Silencing of CUL4 leads to G2-M checkpoint activation that is associated with DNA repair defects.

<p><b>A</b>. Resolution of RAD51 foci was measured upon knockdown of individual Cullins. Schematic of the experiment shown in left. <b>B</b>. RAD51 foci kinetics was performed in cells in which CDT2 was stably knockdown. <b>C</b>. Prior depletion of CDT1 or p21 by siRNAs partially rescues the hype-RAD51 foci formation in MLN4924 treated cells. <b>D</b>. Clonogenic assays were performed for the HEY cells knockdown with CUL4A or CDT2. <b>E</b>. HR repair assays <b>F</b>. NHEJ assay.</p

Model.

<p>UBE2M inhibition impacts DNA damage response and genome integrity involving multiple Cullin ligases.</p