Takeiwa T et al. (2015), Exportin-5 mediates nuclear export of SRP RNA i...

XB-ART-50166

Genes Cells 2015 Apr 01;204:281-91. doi: 10.1111/gtc.12218.

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Exportin-5 mediates nuclear export of SRP RNA in vertebrates.

Takeiwa T , Taniguchi I , Ohno M .

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The signal recognition particle is a ribonucleoprotein complex that is essential for the translocation of nascent proteins into the endoplasmic reticulum. It has been shown that the RNA component (SRP RNA) is exported from the nucleus by CRM1 in the budding yeast. However, how SRP RNA is exported in higher species has been elusive. Here, we show that SRP RNA does not use the CRM1 pathway in Xenopus oocytes. Instead, SRP RNA uses the same export pathway as pre-miRNA and tRNA as showed by cross-competition experiments. Consistently, the recombinant Exportin-5 protein specifically stimulated export of SRP RNA as well as of pre-miRNA and tRNA, whereas an antibody raised against Exportin-5 specifically inhibited export of the same RNA species. Moreover, biotinylated SRP RNA can pull down Exportin-5 but not CRM1 from HeLa cell nuclear extracts in a RanGTP-dependent manner. These results, taken together, strongly suggest that the principal export receptor for SRP RNA in vertebrates is Exportin-5 unlike in the budding yeast.

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Species referenced: Xenopus
Genes referenced: dhfr mmut mt-tr trna xpo1 xpo5 xpot

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	Figure 1. Nuclear export of SRP RNA is independent of CRM1. (A) A mixture of in vitro-transcribed 32P-labeled DHFR mRNA, Xenopus SRP RNA, U1ΔSm RNA, U6Δss RNA and tRNAphe was injected into the nucleus of Xenopus oocytes either with 0.2 μg/oocyte of BSA-NES (lanes 3 and 4) or with the same amount of BSA-mut (mutated NES, lanes 5 and 6). DHFR mRNA and U1ΔSm RNA were m7G-capped, and other RNAs were uncapped. RNA was analyzed immediately (0 h; lanes 1 and 2) or 1.5 h (1.5 h; lanes 3–6) after injection. N, nuclear; C, cytoplasmic fractions. (B) Quantitation of RNA export from four independent experiments as in A. (C) The same as A except that human SRP RNA instead of Xenopus SRP RNA was injected into the nucleus of oocytes either with 0.3 μg/oocyte of BSA-NES or with the same amount of BSA-mut. (D) Quantitation of RNA export from three independent experiments as in C.
	Figure 2. SRP RNA shares the same export pathway with pre-microRNA and tRNA. (A) A mixture of 32P-labeled Xenopus SRP RNA, U1ΔSm RNA, U6Δss RNA and human pre-miR-31 was injected into the nucleus of oocytes either alone (lanes 2 and 3) or with increasing amounts (0.2, 0.5, and 1.0 pmol/oocyte) of unlabeled pre-miR-31 (lanes 4–9). RNA was analyzed immediately (total; lane 1) or 40 min (40 min; lanes 2–9) after injection. (B) The same as A except that 32P-labeled tRNAphe and unlabeled tRNAphe (0.2, 1.0, and 5.0 pmol/oocyte) were used instead of pre-miR-31, and the incubation time was 0 or 30 min. (C) The same as A except that unlabeled U1ΔSm RNA (0.5, 1.0, and 2.0 pmol/oocyte) was used instead of unlabeled pre-miR-31.
	Figure 3. Exportin-5 mediates nuclear export of SRP RNA. (A) A mixture of 32P-labeled Xenopus SRP RNA, U1ΔSm RNA, U6Δss RNA and tRNAphe was injected into the nucleus of oocytes either alone (lanes 2 and 3) or with 150 fmol/oocyte of Exportin-5 (lanes 4 and 5) or Exportin-t (lanes 6 and 7). RNA was analyzed immediately (lane 1) or 15 min (lanes 2–7) after injection. (B) Quantitation of RNA export from the six independent experiments as in A. (C) A mixture of 32P-labeled DHFR mRNA, human SRP RNA, U1ΔSm RNA, U6Δss RNA and tRNAphe was injected either alone (lanes 2 and 3) or with 60 fmol/oocyte of Exportin-5 (lanes 4 and 5) or Exportin-t (lanes 6 and 7). RNA was analyzed immediately (lane 1) or 40 min (lanes 2–7) after injection. (D) Quantitation of RNA export from the three independent experiments in C.
	Figure 4. Endogenous exportin-5 mediates nuclear export of SRP RNA. (A) A mixture of 32P-labeled Xenopus SRP RNA, U1ΔSm RNA, U6Δss RNA, tRNAphe and pre-miR-31 was injected into the nucleus of oocytes either alone (lanes 2 and 3) or with 38 ng/oocyte of anti-Exportin-5 antibody (lanes 4 and 5) or control antibody (lanes 6 and 7). RNA was analyzed immediately (lane 1) or 40 min (lanes 2–7) after injection. (B) Quantitation of RNA export from the four independent experiments as in A. (C) The same as A except that human SRP RNA was used and pre-miR-31 was omitted, and the incubation was 0 or 1.5 h. (D) Quantitation of RNA export from the three independent experiments as in C.
	Figure 5. Exportin-5 interacts with SRP RNA. (A) Biotinylated human SRP RNA was incubated with HeLa cell nuclear extracts (HNE) with or without RanQ69LGTP in buffer A for 30 min at 30°C, and then biotinylated human SRP RNA was pulled down by streptavidin beads. The precipitated proteins were analyzed by Western blotting. (B) The same as A except that the nonbiotinylated RNA competitors were used. (C) The pull-down assay using biotinylated human SRP RNA, the recombinant Exportin-5 and RanQ69LGTP was similarly carried out as A. The precipitated Exportin-5 was analyzed by Western blotting using anti-His tag antibody.

References [+] :

Alavian, Nuclear export of signal recognition particle RNA in mammalian cells. 2004, Pubmed