Aldolase A Enhances Intrahepatic Cholangiocarcinoma Proliferation and Invasion through Promoting Glycolysis.

Energy metabolism reprogramming has been implicated in tumorigenesis and development. Key metabolism enzyme Aldolase A (ALDOA) has been shown to be highly expressed and involved in various kinds of cancers including hepatocellular carcinoma. In this study, we found that ALDOA was highly expressed in clinical intrahepatic cholangiocarcinoma (ICC) tissues, and its high expression was negatively correlated with overall survival (OS) and recurrence-free survival (RFS) in ICC patients. Knockdown of ALDOA expression significantly inhibited the proliferation and migration of ICC both in vitro and in vivo, while highly-expressed ALDOA in ICC cells promoted the proliferation and migration of ICC cells. By applying ALDOA inhibitor and metabolic mass spectrometry tests, we demonstrated that ALDOA modulated the biological characteristics and metabolic level of ICC cells depending on its enzymatic activity. In summary, ALDOA promotes ICC proliferation and migration by enhancing ICC cells glycolysis. Blocking enzymatic activity of ALDOA provides a strategy to inhibit ICC.

Rational design to enhance the catalytic activity of 2-deoxy-D-ribose-5-phosphate aldolase from Pseudomonas syringae pv. syringae B728a.

The 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) enzyme in psychrophilic bacteria has gradually attracted the attention of researchers. A novel gene, deoC (681 bp), encoding DERAPsy, was identified in Pseudomonas syringae pv. syringae B728a, recombinantly expressed in E. coli BL21 and purified via affinity chromatography, which yielded a homodimeric enzyme of 23 kDa. The specific activity of DERAPsy toward 2-deoxy-d-ribose-5-phosphate (DR5P) was 7.37 ± 0.03 U/mg, and 61.32% of its initial activity remained after incubation in 300 mM acetaldehyde at 25 °C for 2 h. Based on the calculation results (dock binding free energy) with the ligand chloroacetaldehyde (CAH), five target substitutions (T16L, F69R, V66K, S188V, and G189R) were identified, in which the DERAPsy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERAPsy. Only the DERAPsy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERAPsy. Fortunately, the aldehyde tolerance of these mutants exhibited no significant decline compared with the wild type. These results indicate an effective strategy for enhancing DERA activity.

Loss of hepatic aldolase B activates Akt and promotes hepatocellular carcinogenesis by destabilizing the Aldob/Akt/PP2A protein complex.

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.

Fructose-Bisphosphate Aldolase A Regulates Hypoxic Adaptation in Hepatocellular Carcinoma and Involved with Tumor Malignancy.

BACKGROUND: Hypoxia is an important factor in malignant tumors, and glycolysis is a major metabolic contributor in their development. Glycolytic enzymes have gained increasing attention as potential therapeutic targets because they are associated with cancer-specific metabolism. Fructose-bisphosphate aldolase A (ALDOA), a key glycolytic enzyme, reportedly is associated with hepatocellular carcinoma (HCC). However, its role in pathogenesis and its clinical significance in HCC remain largely unknown. AIM: To explore the increased expression of ALDOA in HCC in correlation with tumor malignancy, and to investigate the potential regulatory role ALDOA plays in HCC progression through its regulation in hypoxia adaptation. METHODS AND RESULTS: To better understand ALDOA and its correlation with clinicopathological features of HCC, we analyzed 100 HCC clinical specimens using immunohistochemistry analysis. The results show that the ALDOA expression level is significantly higher in advanced HCC and in HCC with venous invasion. Using in vitro knockdown assays, we showed that higher ALDOA expression was positively associated with cell proliferation, cell cycle, apoptosis, and invasion under both normoxic and hypoxic conditions. Evidence shows that the underlying mechanism is due to the regulatory function of ALDOA in glycolysis, the cell cycle, matrix metalloproteinase-mediated extracellular matrix degradation, and epithelial-mesenchymal transformation. CONCLUSIONS: Data indicated that ALDOA is significantly upregulated in HCC tissue and is closely related to HCC malignancy. ALDOA is likely to regulate HCC progression by regulating HCC tumor cell proliferation, apoptosis, and invasion in both normoxic and hypoxic condition.

Protein extract from head-foot tissue of Oncomelania hupensis promotes the growth and development of mother sporocysts of Schistosoma japonicum via upregulation of parasite aldolase gene.

Previous studies showed that protein extract from head-foot tissue of Oncomelania hupensis (O. hupensis) (PhfO), when cocultured with mother sporocysts of Schistosoma japonicum (S. japonicum), was beneficial for parasite's growth and development but the underlying mechanisms remain unclear. One possible strategy for PhfO to promote the growth and development of mother sporocysts of S. japonicum is to upregulate parasite's survival genes. Fructose-1,6-bisphosphate aldolase (ALD), an essential enzyme of glycometabolism in the energy metabolism process, plays an important role in the survival and the growth and development of schistosomes. Using an in vitro coculture system, in this study, we analyzed the potential involvement of the ald gene in the growth and development of mother sporocysts of S. japonicum following coculture with PhfO. We found that coculture with PhfO promoted the growth and development and the survival of mother sporocysts, and increased parasites' ATP consumption level. Mother sporocysts cocultured with PhfO showed a significantly increased expression of the ald gene at both RNA and protein levels. The ALD protein mainly expressed in the cytoplasm of mother sporocysts. Knockdown of ald gene in parasites decreased the ALD protein expression and the ATP consumption level, suppressed the growth and development, and attenuated the survival of mother sporocysts. In ald knockdown mother sporocysts, the effects of PhfO on the ALD expression, the ATP consumption level, the growth and development, and the survival of larvae were significantly abolished. Therefore, the data suggest that PhfO could promote the growth and development, and the survival of mother sporocysts of S. japonicum via upregulating the expression of the ald gene.

Metastasis Suppressor NME1 Directly Activates Transcription of the ALDOC Gene in Melanoma Cells.

BACKGROUND/AIM: NME/NM23 nucleoside diphosphate kinase 1 (NME1) is a metastasis suppressor gene, exhibiting reduced expression in metastatic cancers and the ability to suppress metastatic activity of cancer cells. We previously identified NME1-regulated genes with prognostic value in human melanoma. This study was conducted in melanoma cell lines aiming to elucidate the mechanism through which NME regulates one of these genes, aldolase C (ALDOC). MATERIALS AND METHODS: ALDOC mRNA and protein expression was measured using qRT-PCR and immunoblot analyses. Promoter-luciferase constructs and chromatin immunoprecipitation were employed to measure the impact of NME1 on ALDOC transcription. RESULTS: NME1 enhanced ALDOC transcription, evidenced by increased expression of ALDOC pre-mRNA and activity of an ALDOC promoter-luciferase module. NME1 was detected at the ALDOC promoter, and forced NME1 expression resulted in enhanced occupancy of the promoter by NME1, increased presence of epigenetic activation markers (H3K4me3 and H3K27ac), and recruitment of RNA polymerase II. CONCLUSION: This is the first study to indicate that NME1 induces transcription through its direct binding to the promoter region of a target gene.

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis.

The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth (igr) operon of Mycobacterium tuberculosis, catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of chsH2, i.e., ltp2, was expressed in recombinant Rhodococcus jostii RHA1 in combination with other genes within the igr operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a Km of 6.54 ± 0.90 µM and a kcat of 159 ± 8.50 s-1 ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria.IMPORTANCE The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth (igr) operon of M. tuberculosis, and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice.

Dual effects of fructose on ChREBP and FoxO1/3α are responsible for AldoB up-regulation and vascular remodelling.

Increased production of methylglyoxal (MG) in vascular tissues is one of the causative factors for vascular remodelling in different subtypes of metabolic syndrome, including hypertension and insulin resistance. Fructose-induced up-regulation of aldolase B (AldoB) contributes to increased vascular MG production but the underlying mechanisms are unclear. Serum levels of MG and fructose were determined in diabetic patients with hypertension. MG level had significant positive correlations with blood pressure and fructose level respectively. C57BL/6 mice were fed with control or fructose-enriched diet for 3 months and ultrasonographic and histologic analyses were performed to evaluate arterial structural changes. Fructose-fed mice exhibited hypertension and high levels of serum MG with normal glucose level. Fructose intake increased blood vessel wall thickness and vascular smooth muscle cell (VSMC) proliferation. Western blotting and real-time PCR analysis revealed that AldoB level was significantly increased in both the aorta of fructose-fed mice and the fructose-treated VSMCs, whereas aldolase A (AldoA) expression was not changed. The knockdown of AldoB expression prevented fructose-induced MG overproduction and VSMC proliferation. Moreover, fructose significantly increased carbohydrate-responsive element-binding protein (ChREBP), phosphorylated FoxO1/3α and Akt1 levels. Fructose induced translocation of ChREBP from the cytosol to nucleus and activated AldoB gene expression, which was inhibited by the knockdown of ChREBP. Meanwhile, fructose caused FoxO1/3α shuttling from the nucleus to cytosol and inhibited its binding to AldoB promoter region. Fructose-induced AldoB up-regulation was suppressed by Akt1 inhibitor but enhanced by FoxO1/3α siRNA. Collectively, fructose activates ChREBP and inactivates FoxO1/3α pathways to up-regulate AldoB expression and MG production, leading to vascular remodelling.

Cytosolic proteome profiling of monocytes for male osteoporosis.

In male Caucasians with discordant hip bone mineral density (BMD), we applied the subcellular separation and proteome profiling to investigate the monocytic cytosol. Three BMD-associated proteins (ALDOA, MYH14, and Rap1B) were identified based on multiple omics evidence, and they may influence the pathogenic mechanisms of osteoporosis by regulating the activities of monocytes. INTRODUCTION: Osteoporosis is a serious public health problem, leading to significant mortality not only in aging females but also in males. Peripheral blood monocytes (PBMs) play important roles in bone metabolism by acting as precursors of osteoclasts and producing cytokines important for osteoclast development. The first cytosolic sub-proteome profiling analysis was performed in male PBMs to identify differentially expressed proteins (DEPs) that are associated with BMDs and risk of osteoporosis. METHODS: Here, we conducted a comparative proteomics analysis in PBMs from Caucasian male subjects with discordant hip BMD (29 low BMD vs. 30 high BMD). To decrease the proteome complexity and expand the coverage range of the cellular proteome, we separated the PBM proteome into several subcellular compartments and focused on the cytosolic fractions, which are involved in a wide range of fundamental biochemical processes. RESULTS: Of the total of 3796 detected cytosolic proteins, we identified 16 significant (P < 0.05) and an additional 22 suggestive (P < 0.1) DEPs between samples with low vs. high hip BMDs. Some of the genes for DEPs, including ALDOA, MYH14, and Rap1B, showed an association with BMD in multiple omics studies (proteomic, transcriptomic, and genomic). Further bioinformatics analysis revealed the enrichment of DEPs in functional terms for monocyte proliferation, differentiation, and migration. CONCLUSIONS: The combination strategy of subcellular separation and proteome profiling allows an in-depth and refined investigation into the composition and functions of cytosolic proteome, which may shed light on the monocyte-mediated pathogenic mechanisms of osteoporosis.

ALDOA functions as an oncogene in the highly metastatic pancreatic cancer.

Pancreatic cancer is an aggressive and devastating disease that is characterized by uncontrolled progression, invasiveness and resistance to conventional treatment. In the past decades, much effort has been given to cancer genetics and pathological classification of this disease. Our previous study has uncovered a subgroup of patients with poor outcome, which is characterized by serum signature of CEA(+)/CA125(+)/CA19-9 ≥ 1000 U/mL; however, the underlying biology mechanism remains poorly understood. By using high-throughput screening analysis, we analyzed gene expression signature in highly malignant patients with serum markers of CEA(+)/CA125(+)/CA19-9 ≥ 1000 U/mL. Multiple differentially expressed genes were identified, many of which were closely related with cancer metabolic changes. Treatment of pancreatic cancer cell lines PANC-1 with transforming growth factor-ß (TGF-ß), which was commonly used to induce metastasis, has uncovered that the glycolytic process and antioxidant response was up-regulated upon TGF-ß stimulation. These results were consistent with the high-throughput screening analysis. Subsequent analysis indicated that among glycolytic genes, aldolase A (ALDOA) increased the most significantly upon TGF-ß treatment. Further in vitro and in vivo results demonstrated that ALDOA was associated with proliferation and metastasis of pancreatic cancer cells. Moreover, ALDOA predicted poor prognosis of pancreatic cancer, partially due to its role in E-cadherin expression regulation, and the results were further validated by analysis of the correlation between ALDOA and E-cadherin expression in pancreatic cancer tissue samples. Mechanistically, the role of ALDOA in pancreatic cancer might attribute to its regulation of c-Myc, HIF1α and NRF2 (Nuclear Factor, Erythroid 2-Like 2), which were key regulators of glycolysis and antioxidant response control.

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

OBJECTIVE: To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. METHODS: FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. RESULTS: SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. CONCLUSION: SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. METHODS: We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. RESULTS: ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. CONCLUSION: The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of fructose-1,6-bisphosphate aldolase from Escherichia coli.

Fructose-1,6-bisphosphate aldolase is one of the most important enzymes in the glycolytic pathway and catalyzes the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The full-length fbaB gene encoding fructose-1,6-bisphosphate aldolase class I (FBPA I) was cloned from Escherichia coli strain BL21. FBPA I was overexpressed in E. coli and purified. Biochemical analysis found that the optimum reaction temperature of FBPA I is 330.5 K and that the enzyme has a high temperature tolerance. Crystals of recombinant FBPA I were obtained by the sitting-drop vapour-diffusion technique in a condition consisting of 19 mg ml(-1) FBPA I in 0.1 M Tris pH 9.0, 10%(w/v) polyethylene glycol 8000 and diffracted to 2.0 Šresolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 217.7, b = 114.9, c = 183.9 Å, ß = 124.6°. The asymmetric unit of these crystals may contain ten molecules, giving a Matthews coefficient of 2.48 Å(3) Da(-1) and a solvent content of 50.5%.

Identification and expression of fructose-1,6-bisphosphate aldolase genes and their relations to oil content in developing seeds of tea oil tree (Camellia oleifera).

Tea oil tree (Camellia oleifera, Co) provides a fine edible oil source in China. Tea oil from the seeds is very beneficial to human health. Fructose-1,6-bisphosphate aldolase (FBA) hydrolyzes fructose-1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, two critical metabolites for oil biosynthesis. The objectives of this study were to identify FBA genes and investigate the relationship between FBA gene expression and oil content in developing seeds of tea oil tree. In this paper, four developmentally up-regulated CoFBA genes were identified in Camellia oleifera seeds based on the transcriptome from two seed developmental stages corresponding to the initiation and peak stages of lipid biosynthesis. The expression of CoFBA genes, along with three key oil biosynthesis genes CoACP, CoFAD2 and CoSAD were analyzed in seeds from eight developmental stages by real-time quantitative PCR. The oil content and fatty acid composition were also analyzed. The results showed that CoFBA and CoSAD mRNA levels were well-correlated with oil content whereas CoFAD2 gene expression levels were correlated with fatty acid composition in Camellia seeds. We propose that CoFBA and CoSAD are two important factors for determining tea oil yield because CoFBA gene controls the flux of key intermediates for oil biosynthesis and CoSAD gene controls the synthesis of oleic acid, which accounts for 80% of fatty acids in tea oil. These findings suggest that tea oil yield could be improved by enhanced expression of CoFBA and CoSAD genes in transgenic plants.

Up-regulation of aldolase A and methylglyoxal production in adipocytes.

BACKGROUND AND PURPOSE: We previously reported that up-regulation of aldolase B, a key enzyme in fructose metabolism, was mainly responsible for vascular methylglyoxal (MG) overproduction under different pathological conditions. Here we investigated whether aldolase A, an enzyme of the glycolytic pathway, also caused MG overproduction in insulin-sensitive adipocytes. EXPERIMENTAL APPROACH: The relative contributions of different metabolic pathways or enzymes to MG generation were evaluated in cultured 3T3-L1 adipocytes. KEY RESULTS: Glucose (25 mM) had no effect on aldolase A gene expression, but insulin (100 nM) up-regulated aldolase A mRNA and protein levels in the absence or presence of 25 mM glucose in adipocytes. Treatment with insulin increased levels of basal or glucose (25 mM)-induced MG and glucose 6-phosphate. However, insulin, glucose (25 mM) or their combination had no effect on cellular levels of sorbitol and fructose, but down-regulated gene expression of aldolase B to a similar extent, when compared with the control group. Incubation of 3T3-L1 adipocytes with fructose, acetone, acetol, threonine or glycine (25 mM), with or without insulin did not alter cellular MG levels. The elevated MG levels induced by insulin, glucose (25 mM) or their combination in adipocytes was completely reduced by siRNA knock down of aldolase A or application of 2-deoxy-D-glucose (a non-specific inhibitor of glucose uptake and glycolysis), but not by knock down of aldolase B. CONCLUSION AND IMPLICATIONS: Insulin enhanced MG overproduction in insulin-sensitive adipocytes by up-regulating aldolase A, a mechanism that could be involved in the development of insulin resistance and obesity.

One-pot four-enzyme synthesis of ketoses with fructose 1,6-bisphosphate aldolases from Staphylococcus carnosus and rabbit muscle.

By the action of D-fructose 1,6-bisphosphate aldolases (FruA) from rabbit muscle and Staphylococcus carnosus, various ketoses were synthesized from glyceraldehydes or other aliphatic aldehydes as acceptors in a one-pot four-enzyme system.

The DExD/H box ATPase Dhh1 functions in translational repression, mRNA decay, and processing body dynamics.

Translation, storage, and degradation of messenger ribonucleic acids (mRNAs) are key steps in the posttranscriptional control of gene expression, but how mRNAs transit between these processes remains poorly understood. In this paper, we functionally characterized the DExD/H box adenosine triphosphatase (ATPase) Dhh1, a critical regulator of the cytoplasmic fate of mRNAs. Using mRNA tethering experiments in yeast, we showed that Dhh1 was sufficient to move an mRNA from an active state to translational repression. In actively dividing cells, translational repression was followed by mRNA decay; however, deleting components of the 5'-3' decay pathway uncoupled these processes. Whereas Dhh1's ATPase activity was not required to induce translational inhibition and mRNA decay when directly tethered to an mRNA, ATP hydrolysis regulated processing body dynamics and the release of Dhh1 from these RNA-protein granules. Our results place Dhh1 at the interface of translation and decay controlling whether an mRNA is translated, stored, or decayed.

Compartmentation of the cerebellar nuclei of the mouse.

The cerebellar nuclei integrate inhibitory input from Purkinje cells with excitatory input from mossy and climbing fiber collaterals and are the sole cerebellar output. Numerous studies have shown that the cerebellar cortex is highly compartmentalized into hundreds of genetically determined, reproducible topographic units--transverse zones and parasagittal stripes--that can be identified through the expression patterns of numerous molecules. The Purkinje cell stripes project to the cerebellar nuclei. However, there is no known commensurate topographic complexity in the cerebellar nuclei. Rather, conventional anatomical descriptions identify four major subdivisions--the medial, anterior and posterior interposed, and lateral nuclei--together with a few intranuclear subdivisions. To begin to address the apparent complexity gap, we have used a panel of antigens and transgenes to reveal a reproducible molecular heterogeneity in the mouse cerebellar nuclei. Based on the differential expression patterns, singly and in combination, a new cerebellar nuclear topographic map has been constructed. This reveals the subdivision of the cerebellar nuclei into at least 12 reproducible expression domains. We hypothesize that such heterogeneity is the counterpart of the zones and stripes of the cerebellar cortex.