Conserved Translatome Remodeling in Nematode Species Executing a Shared Developmental Transition

Nematodes of the genus Caenorhabditis enter a developmental diapause state after hatching in the absence of food. To better understand the relative contributions of distinct regulatory modalities to gene expression changes associated with this developmental transition, we characterized genome-wide changes in mRNA abundance and translational efficiency associated with L1 diapause exit in four species using ribosome profiling and mRNA-seq. We found a strong tendency for translational regulation and mRNA abundance processes to act synergistically, together effecting a dramatic remodeling of the gene expression program. While gene-specific differences were observed between species, overall translational dynamics were broadly and functionally conserved. A striking, conserved feature of the response was strong translational suppression of ribosomal protein production during L1 diapause, followed by activation upon resumed development. On a global scale, ribosome footprint abundance changes showed greater similarity between species than changes in mRNA abundance, illustrating a substantial and genome-wide contribution of translational regulation to evolutionary maintenance of stable gene expression.     

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.     

Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells

Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O₂, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.     

Regulated immunoglobulin (Ig) RNA processing does not require specific cis-acting sequences: non-Ig RNA can be alternatively processed in B cells and plasma cells.

Alternative RNA processing of the heavy-chain immunoglobulin mu gene is regulated during B-cell maturation and requires competition between splice and cleavage-polyadenylation reactions that have balanced efficiencies. Studies with modified mu genes have failed to identify gene-specific sequences required for regulation. Thus, the only important feature for regulation may be the balanced competing splice and cleavage-polyadenylation reactions themselves. If this is so, then alternative RNA processing from any gene with similar competitive RNA processing pathways should also be regulated when expression is compared between B cells and plasma cells. To test this prediction, two nonimmunoglobulin genes engineered to have competing splice and cleavage-polyadenylation reactions were expressed in B cells and plasma cells. The ratios of alternative RNAs produced from both genes are different in the two cell types; like the mu gene, relatively more spliced RNA is produced in B cells than in plasma cells. Also, in a survey of mu gene expression in nine non-B-cell lines, only a T-cell line had an expression pattern similar to that of B cells; the expression patterns of all other lines resembled that of the plasma cells. Therefore, regulated mu RNA processing must be mediated by changes in general processing factors whose activity or abundance is regulated, most likely, in B cells.     

Regulation of mRNA translation in stem cells; links to brain disorders

Translational control of gene expression is emerging as a cardinal step in the regulation of protein abundance. Especially for embryonic (ESC) and neuronal stem cells (NSC), regulation of mRNA translation is involved in the maintenance of pluripotency but also differentiation. For neuronal stem cells this regulation is linked to the various neuronal subtypes that arise in the developing brain and is linked to numerous brain disorders. Herein, we review translational control mechanisms in ESCs and NSCs during development and differentiation, and briefly discuss their link to brain disorders.     

Translational control of growth factor and proto-oncogene expression

Control of translation is now understood to be one of the major regulatory events in eukaryotic gene expression. Moreover there is evidence which suggests that aberrant expression of growth-related genes by translational mechanisms makes a significant contribution to cell transformation. However, the mechanisms which regulate translation of specific growth-related mRNAs have yet to be fully elucidated. The majority of these mRNAs have long 5' untranslated regions (UTRs) and three features which are important in translational control have been identified, namely (i) structured regions which inhibit the scanning mechanisms of translation, (ii) regulatory upstream open reading frames and (iii) internal ribosome entry segments which are capable of initiating cap-independent translation. In this review the translational regulation of specific mRNAs encoding growth factors and proto-oncogenes by these three mechanisms will be discussed, together with examples of altered translational regulation in neoplasia.     

Differential regulation of murine B cell Fc gamma RII expression by CD4+ T helper subsets

The murine B cell FcR for IgG (Fc gamma RII) is a membrane glycoprotein reported to mediate inhibition of B cell activation and differentiation. We show that IL-4 inhibits the enhanced expression of Fc gamma RII by LPS-stimulated B cells. This activity is completely reversed by anti-IL-4 mAb and is specific, in that multiple other lymphokines tested do not exert a similar effect. This effect of IL-4 is apparent by day 1 of culture, although maximal inhibition occurs on day 4 at a concentration of 500 U/ml. The IL-4-induced inhibition of enhanced Fc gamma RII expression by LPS stimulation observed on day 4 of culture is associated with a significant reduction in the steady state level of Fc gamma RII beta gene-specific mRNA. IFN-gamma which inhibits many of the effects of IL-4 on B cells, does not reverse the IL-4-induced inhibition of Fc gamma RII membrane expression nor the levels of beta gene-specific mRNA. Fc gamma RII expression is significantly increased in B cells stimulated with antigen-specific, CD4+ T cell clones of the Th1 type (i.e., IL-2 and IFN-gamma-producing). By contrast, three different Th2 clones (i.e., IL-4-producing) fail to stimulate an increase in Fc gamma RII levels. Anti-IL-4 mAb significantly enhanced Fc gamma RII expression by Th2-stimulated B cells indicating that IL-4 was the active, inhibitory, substance produced by the Th2 cells. Supernatants from stimulated Th2 clones inhibited the enhanced expression of Fc gamma RII by LPS-stimulated B cells and this activity was completely reversed by anti-IL-4 mAb. By contrast, supernatants from stimulated Th1 clones further enhanced Fc gamma RII expression by LPS-stimulated B cells. The differential regulation of B cell Fc gamma RII expression by Th subsets may play an important role in the regulation of humoral immunity by altering the sensitivity of B cells to IgG immune complex-mediated inhibition of B cell activation and differentiation in vivo.     

GADD45A protein plays an essential role in active DNA demethylation during terminal osteogenic differentiation of adipose-derived mesenchymal stem cells

Methylation and demethylation of DNA are the complementary processes of epigenetic regulation. These two types of regulation influence a diverse array of cellular activities, including the maintenance of pluripotency and self-renewal in embryonic stem cells. It was generally believed that DNA demethylation occurs passively over several cycles of DNA replication and that active DNA demethylation is rare. Recently, evidence for active DNA demethylation has been obtained in several cancer, neuronal, and embryonic stem cell lines. Studies in embryonic stem cell models, however, suggested that active DNA demethylation might be restricted to the early development of progenitor cells. Whether active demethylation is involved in terminal differentiation of adult stem cells is poorly understood. We provide evidence that active DNA demethylation does occur during terminal specification of stem cells in an adipose-derived mesenchymal stem cell-derived osteogenic differentiation model. The medium CpG regions in promoters of the Dlx5, Runx2, Bglap, and Osterix osteogenic lineage-specific genes were demethylated during the increase in gene expression associated with osteogenic differentiation. The growth arrest and DNA damage-inducible protein GADD45A was up-regulated in these processes. Knockdown of GADD45A led to hypermethylation of Dlx5, Runx2, Bglap, and Osterix promoters, followed by suppression of the expression of these genes and interruption of osteogenic differentiation. These results reveal that GADD45A plays an essential role in gene-specific active DNA demethylation during adult stem cell differentiation. They enhance the current knowledge of osteogenic specification and may also lead to a better understanding of the common mechanisms underlying epigenetic regulation in adult stem cell differentiation.