Palazzo AF et al. (2007), The signal sequence coding region promotes nucl...

XB-ART-37474

PLoS Biol 2007 Dec 01;512:e322. doi: 10.1371/journal.pbio.0050322.

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The signal sequence coding region promotes nuclear export of mRNA.

Palazzo AF , Springer M , Shibata Y , Lee CS , Dias AP , Rapoport TA .

Abstract
In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.

PubMed ID: 18052610
PMC ID: PMC2100149
Article link: PLoS Biol
Grant support: [+]

Species referenced: Xenopus
Genes referenced: ddx39b eif4a3 ins ncbp1 nts

Article Images: [+] show captions

	Figure 1. Microinjected t-ftz mRNA Is Spliced and Translated In Vivo(A) Scheme of the t-ftz transcript. The regions complementary to the oligonucleotides used for FISH (âFISH probeâ) and for RT-PCR (âftz-127Fâ and âftz-444Râ) are indicated. Asterisks represent in-frame stop codons within the intron.(B) The indicated transcripts were translated in vitro using reticulocyte lysate in the presence of 35S-methionine. Samples were separated by SDS-PAGE, and newly synthesized proteins were detected by autoradiography.(C) t-ftz-i was microinjected into either the nuclei (lanes 2â4) or cytoplasm (lane 5) of 20 NIH 3T3 cells, which were then incubated for the indicated times. RT-PCR was performed on the cell extracts using the intron flanking oligonucleotides indicated in (A). The lower band represents the spliced product, and the upper band represents the unspliced product. 18S rRNA was amplified and used as a loading control. Molecular weight markers (M) were loaded in lane 1.(D and E) NIH 3T3 cells were microinjected with t-ftz-i mRNA. FITC-conjugated 70-kDa dextran was co-injected to mark the injected compartment (insets). Cells were incubated for 4 h, fixed and immunostained for either FLAG (D) or HA (E) epitopes and for TRAPÎ±. Overlays of the expressed epitope (green) and TRAPÎ± (red) are shown in the last panel. Scale bar = 10 Î¼m.
	Figure 2. The SSCR Promotes Nuclear Export of mRNA(A and B) NIH 3T3 cells were microinjected with either t-ftz-i (A) or t-ftz-Îi (B) and FITC-conjugated 70-kDa dextran (insets). After the indicated time points, cells were fixed and probed for ftz mRNA by FISH.(C and D) Quantitation of the cytoplasmic/total FISH fluorescence (C) and total FISH fluorescence (D) from cells microinjected with the indicated mRNAs. Each data point represents the average of three experiments, each of which consisted of 15â30 cells. Error bars represent the standard deviation between the three experiments. Relative fluorescence in (D) was calculated by normalizing the total fluorescence at a given time point to the fluorescence at 0 min in each experiment.(E) A plasmid containing the t-ftz-Îi gene, and FITC-conjugated 70-kDa dextran (inset) were microinjected into NIH 3T3 cells. After 30 min, Î±-amanitin was added, and the cells were incubated for an additional 120 min. The cells were fixed and probed for ftz mRNA by FISH.(F) NIH 3T3 cells were microinjected with plasmids containing either the t-ftz-Îi or t-ftz-i genes. After 30 min, the cells were treated with Î±-amanatin and incubated for the indicated time points. The cells were then fixed and probed for ftz mRNA. Nuclear export was quantified as in (C).(G and H) NIH 3T3 cells were microinjected with either c-ftz-Îi (G) or c-ftz-i (H) and FITC-conjugated 70-kDa dextran (insets). After the indicated time points, the cells were fixed, and probed for ftz mRNA by FISH. Scale bar = 15 Î¼m.
	Figure 3. The SSCR Promotes Nuclear Export and ER Targeting of mRNA in COS-7 Cells(A) COS-7 cells were microinjected with t-ftz-Îi mRNA and FITC-conjugated 70-kDa dextran (inset) and then incubated for 90 min. The cells were fixed, probed for ftz mRNA by FISH, and immunostained for TRAPÎ±. An overlay of the mRNA (green) and TRAPÎ± (red) staining is shown in the right panel. Scale bar = 15 Î¼m.(B and C) Quantitation of the cytoplasmic/total FISH fluorescence (C) and total FISH fluorescence (D) in COS-7 cells microinjected with the indicated mRNAs, as described in Figure 2C and 2D.(D) COS-7 cells were transfected with plasmids containing the indicated genes, incubated for 12â18 h, fixed, and probed for ftz mRNA. Cells were then imaged, and the cytoplasmic and nuclear fluorescence was quantified. Each bar represents the average of three experiments in which 20 cells were analyzed. Error bars represent the standard deviation between the three experiments.
	Figure 4. Characterization of SSCR-Mediated Export(A) NIH 3T3 cells were microinjected with t-ftz-Îi mRNA and then incubated in Dulbeco's modified PBS supplemented with either azide or glucose. After the indicated time points, the cells were fixed and probed for ftz mRNA. Quantification of nuclear export was performed as in Figure 2C.(B) NIH 3T3 cells were pre-injected with either wheat germ agglutinin (WGA) or with human IgG (control). After 30 min, the cells were microinjected with t-ftz-Îi mRNA and incubated for the indicated times. The cells were fixed and probed for ftz mRNA. Quantification of nuclear export was performed as in Figure 2C.(C) NIH 3T3 cells were pre-injected with CTE RNA and a marker, cascade blueâconjugated 10-kDa dextran. After 30 min, the cells were microinjected with t-ftz-Îi mRNA and FITC-conjugated 70-kDa dextran. After 1 h, the cells were fixed and probed for ftz mRNA. The arrow indicates a cell pre-injected CTE, which displayed a defect in the export of t-ftz-Îi mRNA. The arrowhead indicates a neighboring cell that was not pre-injected with CTE and showed normal export of t-ftz-Îi mRNA.(D) NIH 3T3 cells were either pre-injected with CTE and marker (CTE) or marker alone (control) and then 30 min later were microinjected with t-ftz-Îi mRNA. At the indicated time points, the cells were fixed and probed for ftz mRNA. Quantification of nuclear export was performed as in Figure 2C.(E) HeLa cells were treated with siRNA oligonucleotides directed against both UAP56 and URH49 or with control oligonucleotides (Mock). Cell lysates were collected and separated by SDS-PAGE. UAP56 and CBP80 were detected by Western blotting.(F and G) HeLa cells were treated for 24 h with siRNA against both UAP56 and URH49 (F and G) or for 48 h against eIF4AIII (G). The cells were then microinjected with either t-ftz-Îi, t-ftz-i, or c-ftz-i mRNA and FITC-conjugated 70-kDa dextran (insets). After 1 h, the cells were fixed, probed for ftz mRNA, imaged (F), and quantified for nuclear export (G). Each bar represents the average of three experiments, in which 20â30 cells were analyzed. Error bars represent the standard deviation between the three experiments. Scale bar = 15 Î¼m.(H and I) Quantification of nuclear export and stability of microinjected t-ftz-Îi or c-ftz-i mRNA capped with either a natural cap (mGpppG), trimethylated cap (3mGpppG), or adenine cap (ApppG). At the indicated time points, the microinjected cells were fixed and probed for ftz mRNA. Quantitation of nuclear export (H) and of the total fluorescence (I) was performed as in Figure 2C and 2D, respectively.
	Figure 5. The SSRC Is an RNA Element That Promotes Nuclear Export(A and B) COS-7 cells were microinjected with either 3R-ftz-Îi, fs-ftz-Îi, UUG-ftz-Îi, or t-ftz-Îi mRNA and FITC-conjugated 70-kDa dextran (insets) and then incubated for 60 min (A) or the indicated time points (B). Cells were fixed and probed for ftz mRNA. Quantitation of the nuclear export (B) was performed as in Figure 2C.(CâE) COS-7 cells were pretreated with either pactamycin or DMSO (control) for 20 min and then microinjected with t-ftz-Îi mRNA. After 1 h the cells were fixed and probed for ftz mRNA (C). Quantitation of the nuclear export (D) and stability (E) were carried out as in Figure 2C and 2D, except that the relative fluorescence (E) was calculated by normalizing the fluorescence to that of the control treated cells. Scale bar = 15 Î¼m.
	Figure 6. Large-Scale Genomic Analysis of the SSCRAll analysis was performed on four data sets from each genome; the first 69 nucleotides (nts) after the start codon of SSCR-containing ORFs (red bars), the middle 69 nts of SSCR-containing ORFs (yellow bars), the first 69 nts from ORFs lacking a SSCR (dark blue bars), and the middle 69 nts from this set of ORFs (light blue). Each bar represents the average across the entire set of sequences within a given genome.(A and B) For each sequence the overall percentage of adenine (A) and the longest tract lacking an adenine (no-A-tract) (B) was calculated for each ORF and then averaged over each set.(C and D) The average ratio of encoded leucines to isoleucines (C), and encoded arginines to lysines (D) was calculated.(E and F) The average ratio of [CUU, CUC, CUG, UUG]/[CUA, UUA] codons for leucine (E), and [UCU, UCC, UCG]/[UCA, AGU, AGC] codons for serine (F) was calculated.(G) The codons for leucine, valine, serine, proline, alanine, argenine, and glycine were compiled, and the average ratio between the adenine-lacking codons to adenine-containing codons was calculated.
	Figure 7. Silent Adenine Mutations Inhibit SSCR-Dependent Nuclear Export of mRNA(AâC) COS-7 cells were microinjected with t-ftz transcripts containing seven silent adenine mutations within the SSCR. FITC-conjugated 70-kDa dextran was co-injected as a marker (insets). Cells were incubated for 1 h (A) or various time points (B and C) to allow for export, then fixed and probed for ftz mRNA (A). Quantification of nuclear export (B) and stability (C) were performed as in Figure 2C and 2D.(DâF) As in (AâC), except that COS-7 cells were microinjected with either wild-type insulin transcripts or an insulin transcript containing five silent adenine mutations within the SSCR (5A-insulin). FITC-conjugated 70-kDa dextran was co-injected (insets). Cells were incubated for 1 h (D) or various time points (E and F) to allow for export, then fixed and probed for ftz mRNA (D). Quantification of nuclear export (E) and stability (F) as in Figure 2C and 2D. Scale bar = 15 Î¼m.(G and H) COS-7 cells were microinjected with plasmids containing the indicated genes. After 30 min, the cells were treated with Î±-amanatin and incubated for the indicated time points. The cells were then fixed and probed for ftz mRNA. Quantification of nuclear export (G) and stability (H) were performed as in Figure 2C and 2D, except that each data point represents the average of four experiments, each of which consisted of 20â40 cells. Error bars represent the standard deviation between the four experiments.

References [+] :

Alcázar-Román, Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export. 2006, Pubmed