Limits...
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.

Show MeSH
The SSRC Is an RNA Element That Promotes Nuclear Export(A and B) COS-7 cells were microinjected with either 3R-ftz-Δi, fs-ftz-Δi, UUG-ftz-Δi, or t-ftz-Δi 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.(C–E) COS-7 cells were pretreated with either pactamycin or DMSO (control) for 20 min and then microinjected with t-ftz-Δi 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 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2100149&req=5

pbio-0050322-g005: The SSRC Is an RNA Element That Promotes Nuclear Export(A and B) COS-7 cells were microinjected with either 3R-ftz-Δi, fs-ftz-Δi, UUG-ftz-Δi, or t-ftz-Δi 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.(C–E) COS-7 cells were pretreated with either pactamycin or DMSO (control) for 20 min and then microinjected with t-ftz-Δi 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 μm.

Mentions: In principle, the SSCR nuclear export signal could either be an RNA element or its translated amino acid sequence, the signal sequence. To distinguish between these possibilities, we altered five nucleotides within the SSCR such that three encoded hydrophobic residues within the core of the signal sequence were changed to arginines (3R-ftz, see Figure S3). The mutated signal sequence should no longer target the translation product to the ER membrane. Upon injection into the nuclei of COS-7 cells, 3R-ftz-Δi mRNA was exported with a kinetics similar to that of t-ftz-Δi (Figure 5A; quantitation in Figure 5B). As expected, the mRNA was no longer targeted to the ER, and instead was distributed diffusely in the cytoplasm (Figure 5A). Its translation product was targeted to mitochondria (unpublished data), possibly because the signal sequence was converted into an amphipatic helix typical of mitochondrial targeting sequences [48].


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)

The SSRC Is an RNA Element That Promotes Nuclear Export(A and B) COS-7 cells were microinjected with either 3R-ftz-Δi, fs-ftz-Δi, UUG-ftz-Δi, or t-ftz-Δi 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.(C–E) COS-7 cells were pretreated with either pactamycin or DMSO (control) for 20 min and then microinjected with t-ftz-Δi 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 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100149&req=5

pbio-0050322-g005: The SSRC Is an RNA Element That Promotes Nuclear Export(A and B) COS-7 cells were microinjected with either 3R-ftz-Δi, fs-ftz-Δi, UUG-ftz-Δi, or t-ftz-Δi 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.(C–E) COS-7 cells were pretreated with either pactamycin or DMSO (control) for 20 min and then microinjected with t-ftz-Δi 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 μm.
Mentions: In principle, the SSCR nuclear export signal could either be an RNA element or its translated amino acid sequence, the signal sequence. To distinguish between these possibilities, we altered five nucleotides within the SSCR such that three encoded hydrophobic residues within the core of the signal sequence were changed to arginines (3R-ftz, see Figure S3). The mutated signal sequence should no longer target the translation product to the ER membrane. Upon injection into the nuclei of COS-7 cells, 3R-ftz-Δi mRNA was exported with a kinetics similar to that of t-ftz-Δi (Figure 5A; quantitation in Figure 5B). As expected, the mRNA was no longer targeted to the ER, and instead was distributed diffusely in the cytoplasm (Figure 5A). Its translation product was targeted to mitochondria (unpublished data), possibly because the signal sequence was converted into an amphipatic helix typical of mitochondrial targeting sequences [48].

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