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SIRT7 is an NAD + -dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-ribosomal RNA (rRNA) and promotes rRNA synthesis. Here we show that SIRT7 is also associated with small nucleolar RNP (snoRNPs) that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of pre-rRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. SIRT7 is a protein deacetylase with important roles in rRNA synthesis, ribosome biogenesis and cell proliferation. Here the authors show a role of SIRT7 in rRNA maturation via deacetylation of U3-55k, a core component of the U3 snoRNP complex.

AMP-activated protein kinase (AMPK) senses changes in the intracellular AMP/ATP ratio, switching off energy-consuming processes and switching on catabolic pathways in response to energy depletion. Here, we show that AMPK down-regulates rRNA synthesis under glucose restriction by phosphorylating the RNA polymerase I (Pol I)-associated transcription factor TIF-IA at a single serine residue (Ser-635). Phosphorylation by AMPK impairs the interaction of TIF-IA with the TBP-containing promoter selectivity factor SL1, thereby precluding the assembly of functional transcription initiation complexes. Mutation of Ser-635 compromises down-regulation of Pol I transcription in response to low energy supply, supporting that activation of AMPK adapts rRNA synthesis to nutrient availability and the cellular energy status.

Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF(I)110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD(+)-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF(I)110 is prevented. The results demonstrate that reversible phosphorylation of TAF(I)110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF‐I. In exploring the functional significance of the promoter‐proximal terminator, we found that TTF‐I associates with the p300/CBP‐associated factor PCAF, suggesting that TTF‐I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF I 68, the second largest subunit of the TATA box‐binding protein (TBP)‐containing factor TIF‐IB/SL1, and acetylation enhances binding of TAF I 68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF‐IB/SL1 being required for rDNA transcription, the NAD + ‐dependent histone deacetylase mSir2a deacetyl ates TAF I 68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF‐IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein–protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference‐mediated knockdown of NM1 and WSTF reduced pre‐rRNA synthesis in vivo , and antibodies to WSTF inhibited Pol I transcription on pre‐assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G1-specific cyclin-dependent kinase (cdk)/cyclin complexes. After G1progression UBF is phosphorylated at serine 388 by cdk2/cyclin E and cdk2/cyclin A. Conversion of serine 388 to glycine abolishes UBF activity, whereas substitution by aspartate enhances the transactivating function of UBF. Protein-protein interaction studies reveal that phosphorylation at serine 388 is required for the interaction between RNA polymerase I and UBF. The results suggest that phosphorylation of UBF represents a powerful means of modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent fashion.

Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (${\\rm DEDD}^{-/-}$) embryonic fibroblasts is increased compared with that in ${\\rm DEDD}^{+/+}$ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G₂/M stage. Interestingly, ${\\rm DEDD}^{-/-}$ cells also demonstrated decreased G₁ duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, ${\\rm DEDD}^{-/-}$ mice show decreased body and organ weights relative to ${\\rm DEDD}^{+/+}$ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.

Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14APD) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14APD cells. hCDC14APD cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14APD cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast.

Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAFI110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD+-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAFI110 is prevented. The results demonstrate that reversible phosphorylation of TAFI110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.