Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia.

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.

Multilevel regulation of HIF-1 signaling by TTP.

Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.

Mutation in a primate-conserved retrotransposon reveals a noncoding RNA as a mediator of infantile encephalopathy.

The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. Here we report homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. We show here that a mutation in a unique transposable element-containing RNA is associated with lethal encephalopathy, and we suggest that RNAs that harbor evolutionarily recent repetitive elements may play important roles in human brain development.

Phorbol-Ester Mediated Suppression of hASH1 Synthesis: Multiple Ways to Keep the Level Down.

Human achaete-scute homolog-1 (hASH1), encoded by the human ASCL1 gene, belongs to the family of basic helix-loop-helix transcription factors. hASH1 and its mammalian homolog Mash1 are expressed in the central and peripheral nervous system during development, and promote early neuronal differentiation. Furthermore, hASH1 is involved in the specification of neuronal subtype identities. Misexpression of the transcription factor is correlated with a variety of tumors, including lung cancer and neuroendocrine tumors. To gain insights into the molecular mechanisms of hASH1 regulation, we screened for conditions causing changes in hASH1 gene expression rate. We found that treatment of human neuroblastoma-derived Kelly cells with phorbol 12-myristate 13-acetate (PMA) resulted in a fast, strong and long-lasting suppression of hASH1 synthesis. Reporter gene assays with constructs, in which the luciferase activity was controlled either by the ASCL1 promoter or by the hASH1 mRNA untranslated regions (UTRs), revealed a mainly UTR-dependent mechanism. The hASH1 promoter activity was decreased only after 48 h of PMA administration. Our data indicate that different mechanisms acting consecutively at the transcriptional and post-transcriptional level are responsible for hASH1 suppression after PMA treatment. We provide evidence that short term inhibition of hASH1 synthesis is attributed to hASH1 mRNA destabilization, which seems to depend mainly on protein kinase C activity. Under prolonged conditions (48 h), hASH1 suppression is mediated by decreased promoter activity and inhibition of mRNA translation.

Translational regulation of the human achaete-scute homologue-1 by fragile X mental retardation protein.

Fragile X syndrome is a common inherited cause of mental retardation that results from loss or mutation of the fragile X mental retardation protein (FMRP). In this study, we identified the mRNA of the basic helix-loop-helix transcription factor human achaete-scute homologue-1 (hASH1 or ASCL1), which is required for normal development of the nervous system and has been implicated in the formation of neuroendocrine tumors, as a new FMRP target. Using a double-immunofluorescent staining technique we detected an overlapping pattern of both proteins in the hippocampus, temporal cortex, subventricular zone, and cerebellum of newborn rats. Forced expression of FMRP and gene silencing by small interference RNA transfection revealed a positive correlation between the cellular protein levels of FMRP and hASH1. A luciferase reporter construct containing the 5'-untranslated region of hASH1 mRNA was activated by the full-length FMRP, but not by naturally occurring truncated FMR proteins, in transient co-transfections. The responsible cis-element was mapped by UV-cross-linking experiments and reporter mutagenesis assays to a (U)(10) sequence located in the 5'-untranslated region of the hASH1 mRNA. Sucrose density gradient centrifugation revealed that hASH1 transcripts were translocated into a translationally active polysomal fraction upon transient transfection of HEK293 cells with FMRP, thus indicating translational activation of hASH1 mRNA. In conclusion, we identified hASH1 as a novel downstream target of FMRP. Improved translation efficiency of hASH1 mRNA by FMRP may represent an important regulatory switch in neuronal differentiation.