Alan M. Tartakoff

Dbp5p, a cytosolic RNA helicase, is required for poly(A) + RNA export

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP‐dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Mechanisms of mammalian iron homeostasis

Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in the acquisition or distribution of the metal causes anemia, whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways as well as in mechanisms underlying intracellular iron trafficking, an important but less studied area of mammalian iron homeostasis.

Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.

The RCC1 gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae (MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature‐sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtr1/prp20/srm1) mutants accumulate nuclear poly(A)+ RNA at 37 degrees C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37 degrees C. Nevertheless, studies with the corresponding BHK‐21 cell mutant indicate that protein import into the nucleus continues. MTR1 homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A)+ RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCC1p acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNR1 is essential, but both code for proteins extremely similar to Ran and can suppress mtr1 mutations in allele‐specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes.

Visual Experience Regulates Transient Expression and Dendritic Localization of Fragile X Mental Retardation Protein

Fragile X syndrome is the most common form of inherited mental retardation and is caused by the loss of function of the Fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein thought to play a key role in protein synthesis-dependent synaptic plasticity. The regulation of FMRP expression itself is also likely to be an important control point in this process. Here we used dark-reared/light-exposed rats to determine the role of experience in regulating FMRP levels in the visual cortex. We find that FMRP levels increase in the cell bodies and dendrites of visual cortical neurons after as little as 15 min of light exposure. Remarkably, FMRP expression in these neurons returns to baseline levels by 30 min of light exposure. These changes were post-transcriptional because the FMR1 mRNA levels remained constant over this time period. A transient increase in FMRP levels was also observed in synaptic fractions prepared from visual cortices of light-exposed animals. In contrast, α-calcium/calmodulin-dependent kinase II expression showed a sustained upregulation under these conditions. Finally, the increase in FMRP expression was inhibited by blockade of NMDA receptors. This tight temporal-spatial regulation suggests that FMRP plays a dynamic role in a distinct epoch of experience-dependent synaptic plasticity.

The Arrest of Secretion Response in Yeast: Signaling from the Secretory Path to the Nucleus via Wsc Proteins and Pkc1p

The arrest of secretion response (ASR) in sec mutants reversibly inhibits nuclear import and relocates nuclear proteins to the cytoplasm. sec mutants also relocate nucleoporins; however, endocytic and Golgi-to-vacuole transport mutants do not cause relocation. The ASR requires Wsc membrane proteins that are trapped along the secretory path, rather than those which are at the plasma membrane. The activity of the downstream kinase, Pkc1p, is also required; however, the Pkc1p MAP kinase cascade is not. sec mutants initiate compensatory transcriptional changes distinct from those of the unfolded protein response.

Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export.

Heat shock causes major positive and negative changes in gene expression, drastically alters the appearance of the nucleolus and inhibits rRNA synthesis. We here show that it causes many yeast nucleolar proteins, including the fibrillarin homolog Nop1p, to relocate to the cytoplasm. Relocation depends on several proteins implicated in mRNA transport (Mtrps) and is reversible. Two observations indicate, surprisingly, that disassembly results from a reduction in Ssa protein (Hsp70) levels: (i) selective depletion of Ssa1p leads to disassembly of the nucleolus; (ii) preincubation at 37 degrees C protects the nucleolus against disassembly by heat shock, unless expression of Ssa proteins is specifically inhibited. We observed that heat shock or reduction of Ssa1p levels inhibits protein import into the nucleus and therefore we propose that inhibition of import leads to disassembly of the nucleolus. These observations provide a simple explanation of the effects of heat shock on the anatomy of the nucleolus and rRNA transcription. They also extend understanding of the path of nuclear export. Since a number of nucleoplasmic proteins also relocate upon heat shock, these observations can provide a general mechanism for regulation of gene expression. Relocation of the hnRNP‐like protein Mtr13p (= Npl3p, Nop3p), explains the heat shock sensitivity of export of average poly(A)+ RNA. Strikingly, Hsp mRNA export appears not to be affected.

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

Hydrophilic anchor-deficient Thy-1 is secreted by a class E mutant T lymphoma

To investigate the mechanism of glycophospholipid anchoring of the surface antigen Thy-1, we have undertaken a comparative biosynthetic study using a wild-type Thy-1+ murine T lymphoma (BW5147) and a mutant T lymphoma (class E) that synthesizes Thy-1 but fails to express it on the plasma membrane. Labelling experiments with D-[2-3H]mannose demonstrate that, unlike the wild type, the mutant cells are secreting large amounts of Thy-1 and that the secreted molecules are hydrophilic. Moreover, unlike the wild type, they fail to incorporate [3H]palmitic acid into Thy-1. Both wild-type and mutant cells do incorporate labeled galactose and fucose into Thy-1. We conclude that the lack of surface expression of Thy-1 by this mutant results from the failure to add anchor components to Thy-1.

How to make a glycoinositol phospholipid anchor

Essentially all eukaryotic cells express proteins on their surface that are anchored by a glycoinositol phospholipid. This anchor moiety may endow such proteins with unusual properties. The definition of the biosynthetic path that constructs these anchors is now in its final stages. Mutations that interrupt this path are, remarkably, compatible with survival of cells in culture, but are associated with at least one human disease.