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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 - PLoS Biol. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.

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.

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Related in: MedlinePlus

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 (“FISH probe”) and for RT-PCR (“ftz-127F” and “ftz-444R”) 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 2–4) 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 TRAPα. Overlays of the expressed epitope (green) and TRAPα (red) are shown in the last panel. Scale bar = 10 μm.
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pbio-0050322-g001: 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 (“FISH probe”) and for RT-PCR (“ftz-127F” and “ftz-444R”) 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 2–4) 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 TRAPα. Overlays of the expressed epitope (green) and TRAPα (red) are shown in the last panel. Scale bar = 10 μm.

Mentions: To study the nuclear export of mRNA coding for a secretory protein, we used a model mRNA that is derived from a fragment of the fushi tarazu (ftz) gene. The original construct contains an intron and was previously used to monitor mRNA splicing and nuclear export in Xenopus oocytes [1,34]. The construct was modified by adding a Kozak consensus sequence to allow efficient expression in mammalian cells. Sequences encoding FLAG and hemagglutinin (HA) epitopes were included at the 5′ and 3′ ends of the open reading frame (ORF), respectively, to monitor translation of the mRNA. Because the intron contains in-frame stop codons (Figure 1A; asterisks), the HA epitope will only be synthesized if the mRNA is spliced. To target the translation product to the ER, we attached an SSCR derived from the mouse major histocompatibility complex (MHC) class 2 molecule H2-K1. The final construct is called t-ftz-i (Figure 1A and Figure S1).


The signal sequence coding region promotes nuclear export of mRNA.

Palazzo AF, Springer M, Shibata Y, Lee CS, Dias AP, Rapoport TA - PLoS Biol. (2007)

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 (“FISH probe”) and for RT-PCR (“ftz-127F” and “ftz-444R”) 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 2–4) 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 TRAPα. Overlays of the expressed epitope (green) and TRAPα (red) are shown in the last panel. Scale bar = 10 μm.
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Related In: Results  -  Collection

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pbio-0050322-g001: 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 (“FISH probe”) and for RT-PCR (“ftz-127F” and “ftz-444R”) 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 2–4) 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 TRAPα. Overlays of the expressed epitope (green) and TRAPα (red) are shown in the last panel. Scale bar = 10 μm.
Mentions: To study the nuclear export of mRNA coding for a secretory protein, we used a model mRNA that is derived from a fragment of the fushi tarazu (ftz) gene. The original construct contains an intron and was previously used to monitor mRNA splicing and nuclear export in Xenopus oocytes [1,34]. The construct was modified by adding a Kozak consensus sequence to allow efficient expression in mammalian cells. Sequences encoding FLAG and hemagglutinin (HA) epitopes were included at the 5′ and 3′ ends of the open reading frame (ORF), respectively, to monitor translation of the mRNA. Because the intron contains in-frame stop codons (Figure 1A; asterisks), the HA epitope will only be synthesized if the mRNA is spliced. To target the translation product to the ER, we attached an SSCR derived from the mouse major histocompatibility complex (MHC) class 2 molecule H2-K1. The final construct is called t-ftz-i (Figure 1A and Figure S1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.

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.

Show MeSH
Related in: MedlinePlus