p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum

The localization of many secretory mRNAs to the endoplasmic reticulum does not require ribosomes or translation, but is instead promoted by p180, an abundant, membrane-bound protein that likely binds directly to mRNA

Molecular Characterization of Chinese Hamster Cells Mutants Affected in Adenosine Kinase and Showing Novel Genetic and Biochemical Characteristics

<p>Abstract</p> <p>Background</p> <p>Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms.</p> <p>Results</p> <p>We show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg²⁺and phosphate ions is essential for AdK function. Another mutant, Fom^R-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser₁₉₁Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the Fom^R-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins.</p> <p>Conclusions</p> <p>These AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.</p

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data

RanBP2/Nup358 Potentiates the Translation of a Subset of mRNAs Encoding Secretory Proteins

In higher eukaryotes, most mRNAs that encode secreted or membrane-bound proteins contain elements that promote an alternative mRNA nuclear export (ALREX) pathway. Here we report that ALREX-promoting elements also potentiate translation in the presence of upstream nuclear factors. These RNA elements interact directly with, and likely co-evolved with, the zinc finger repeats of RanBP2/Nup358, which is present on the cytoplasmic face of the nuclear pore. Finally we show that RanBP2/Nup358 is not only required for the stimulation of translation by ALREX-promoting elements, but is also required for the efficient global synthesis of proteins targeted to the endoplasmic reticulum (ER) and likely the mitochondria. Thus upon the completion of export, mRNAs containing ALREX-elements likely interact with RanBP2/Nup358, and this step is required for the efficient translation of these mRNAs in the cytoplasm. ALREX-elements thus act as nucleotide platforms to coordinate various steps of post-transcriptional regulation for the majority of mRNAs that encode secreted proteins

<i>ALPP</i> and <i>CALR</i>, but not <i>t-ftz</i> or <i>INSL3</i>, mRNA remain associated with the ER independently of ribosomes and translation.

<p>(A–E) COS-7 cells were transfected with plasmids containing either the <i>t-ftz</i> (A), <i>INSL3</i> (A–B), <i>ALPP</i> (A, C), <i>cyto-ALPP</i> (a version of <i>ALPP</i> lacking signal sequence and transmembrane domain coding regions; A, D–E), or <i>CALR</i> (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused <i>INSL3</i> mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing <i>cyto-ALPP</i> mRNA. <i>cyto-ALPP</i> mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of <i>cyto-ALPP</i> mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.</p

Proteins enriched in ERMAP fraction.

<p>List of significantly enriched proteins (<i>p</i><0.05) in the ERMAP fraction (“RNase+”, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001336#pbio-1001336-g005" target="_blank">Figure 5</a>, lane 9) compared to the control sample (“RNase−”, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001336#pbio-1001336-g005" target="_blank">Figure 5</a>, lane 10) as analyzed by mass spectrometry (for a larger list, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001336#pbio.1001336.s008" target="_blank">Table S1</a>). Included in the table are the Entrez Gene ID, average number (“AVG”), and standard deviation (“STD”) of peptides from the analyses performed on three independent experiments. In addition the average number of peptides from all the components of the MSC was also tabulated, some of which <i>p</i>>0.05. The <i>p</i> values were determined using a paired two-tailed Student <i>t</i> test. On average, 2,427±311 total peptides were recovered from the RNase+ samples, and 1,684±266 total peptides were recovered from the RNase− samples.</p>a<p>rRNA or snRNA binding protein.</p>b<p>Primarily involved in DNA binding but has been reported to bind to RNA.</p>c<p>Members of the MSC where <i>p</i>>0.05.</p

p180 is required for the ER-association of mRNA.

<p>(A–H) U2OS cells were infected with specific shRNAs against p180 (shRNA clones B9 and B10), kinectin or CLIMP63, or with control lentivirus (“Cont”). (A–B) Cell lysates were separated by SDS-PAGE and immunoblotted for p180, CLIMP63, kinectin, αtubulin, Trapα, and Sec61β. (C–E) Cells depleted of p180 (Clone B9; C, E) or kinectin (D), or infected with control lentivirus (“cont’; C–E), were treated with control media (no drug, “ND”) or HHT for 30 min, then extracted with digitonin and stained for poly(A) mRNA using poly(dT) FISH probes. (C–D) For each cell the total level of ER-associated poly (A) FISH signal (normalized from the background (0), to the brightest cell in the entire experiment (1); <i>y</i>-axis) was plotted against cell size (pixels squared, <i>x</i>-axis). For each data set a regression line was plotted and the coefficient of determination (R²) was indicated. (E) The ratio of ER to nuclear poly(A) fluorescence was quantified and normalized. Each bar represents the average and standard error of five independent experiments, each consisting of the average of >30 cells. (F–H) Cells were depleted of p180 or kinectin with specific shRNAs, or infected with control lentivirus (“Cont”), then transfected with plasmids containing either the <i>ALPP</i> (F–G) or <i>CALR</i> (H) gene. The cells were allowed to express mRNA for 18–24 h, then treated with control media (no drug, “ND”) or HHT for 30 min, and then extracted with digitonin. Cells were then fixed, stained for mRNA using specific FISH probes against the exogenous mRNA, and imaged. Nuclei are outlined with blue dotted lines. Scale bar = 20 µm. (G–H) The fluorescence intensity on the ER and nucleus were quantified. Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background.</p

Identification of proteins that associate with ER-derived mRNAs.

<p>Cycloheximide-treated U2OS cells were digitonin extracted and centrifuged at low speed to separate cytoplasm (supernatant, lane 2) from ER and nuclear components (pellet, lane 3). The pellet was then extracted with Triton-X100 and centrifuged at low speed to separate ER (supernatant, lane 4) from the nucleus (pellet). Note that the ER fraction is relatively free of histones (“H”), which are found in the ER+Nuc fraction (lane 2). The solubilized ER fraction was then subjected to high-speed centrifugation through a sucrose cushion to separate polysomes (pellet, lane 6) from the rest of the ER (supernatant, lane 5). The polysomes were then treated with either RNase A (lanes 7, 9) or control buffer (lanes 8, 10) at 37°C for 15 min to digest all mRNA. The samples were then subjected to another high-speed centrifugation step to separate ribosomes and associated proteins (pellet, lanes 7–8) from proteins released by RNase A (supernatant, lane 9) or control treatments (supernatant, lane 10). Note that the treatments did not release ribosomal proteins (“R”), which all remained in the pellets (lanes 7,8). All fractions were separated on a 4%–20% gradient SDS-PAGE and visualized by Coomassie blue staining. To estimate protein sizes, molecular weight markers (MWM) were loaded (lane 1, sizes of each band in kD are indicated on the left). Lanes 9 and 10 were cut and sent for mass spectrometry analysis.</p