Izumi H et al. (2014), p54nrb/NonO and PSF promote U snRNA nuclear exp...

XB-ART-53412

Nucleic Acids Res 2014 Apr 01;426:3998-4007. doi: 10.1093/nar/gkt1365.

Show Gene links Show Anatomy links

p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly.

Izumi H , McCloskey A , Shinmyozu K , Ohno M .

???displayArticle.abstract???
The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m(7)G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.

???displayArticle.pubmedLink??? 24413662
???displayArticle.pmcLink??? PMC3973303
???displayArticle.link??? Nucleic Acids Res

Species referenced: Xenopus
Genes referenced: csnk2b dhfr hnrnpc isyna1 nono phax ran sfpq xpo1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Characterization of the activity that stimulates PHAX-recruitment to RNA substrates. (A) A mixture of 32P-labeled in vitro transcribed RNAs containing A-capped DHFR mRNA, m7G-capped Ftz intronless mRNAs (15), A-capped elongated U1ΔSm RNA [U1 + 50 RNAs, (15)] and m7G-capped U1ΔSm RNA was mixed with CBC and GST-PHAX or GST (0.2 µM each) and a GST pull-down assay was performed. Co-precipitated RNA was analyzed by 8% denaturating PAGE followed by autoradiography. HNE was added to final concentration of 0–5% in lanes 2–4. Quantification of U1RNA bands in the pull-down panels is shown. (B) The final Mono S fractions were analyzed by 4–20% gradient SDS/PAGE and silver staining. An index for the activity is shown on the top. (C–E) A GST pull-down assay as in (A) in the absence or presence of recombinant p54nrb, PSF or HNE (15%) as indicated. Quantification of U1RNA bands in the pull-down panels is shown. (F) HeLa cells were transfected with siRNAs against p54nrb and PSF and western blotting was performed with antibodies directed against PSF, p54nrb or CBP20 (loading control). (G) A GST pull-down assay as in (A) in the absence or presence of HNEs from HeLa cells treated with siRNAs against p54nrb and PSF or with control siRNA, with or without recombinant p54nrb and PSF as indicated. Quantification of U1RNA bands in the pull-down panels is shown. Concentrations of p54nrb and PSF in the corresponding HNEs as measured by western blotting are shown on the bottom.
	Figure 2. p54nrb and PSF interact with PHAX and stimulate the formation of the pre-complex. (A–C) A 32P-labeled capped U1ΔSm RNA probe was incubated for 30 min at 25°C in the presence or absence of recombinant CBC, PHAX, p54nrb and PSF as indicated. The samples were fractionated by native 6% PAGE followed by autoradiography. Free RNA probe and major complexes are indicated on the left. (D) and (E) GST-PHAX or GST (0.2 µM each) was mixed with p54nrb (D) or PSF (E) in the absence or presence of RNase A and a GST-pulldown was performed. The precipitated protein was analyzed by western blotting. (F) Flag-tagged PHAX was transiently expressed in HeLa cells. A flag IP was performed and the precipitated protein was analysed by western blotting.
	Figure 3. p54nrb and PSF interact with CRM1 and Ran, and stimulate the formation of the U snRNA export complex. (A) Recombinant p54nrb, PSF and hnRNP A1 were mixed with recombinant CRM1 or RanGTP. IPs were performed with the indicated antibodies and the precipitated protein was analyzed by western blotting. (B) A 32P-labeled capped U1ΔSm RNA probe was incubated for 30 min at 25°C in the presence or absence of CBC, phosphorylated PHAX and/or CRM1 plus RanQ69LGTP as indicated. PHAX was phosphorylated by recombinant kinase CK2 (7). The samples were fractionated by native 6% PAGE followed by autoradiography. Free RNA probe and major complexes are indicated on the sides.
	Figure 4. p54nrb and PSF are involved in U snRNA export in living cells. (A) A mixture of 32 P-labeled m7G-capped DHFR mRNA, m7G-capped U1ΔSm, m7G-capped U2ΔSm, m7G-capped U5ΔSm and U6Δss RNAs was injected into the nucleus of Xenopus oocytes either alone or together with recombinant human p54nrb and PSF (400 fmol/oocyte). RNA was extracted from nuclear (N) and cytoplasmic (C) fractions, immediately (0 min) or 60 min after microinjection and analyzed by 8% denaturating PAGE followed by autoradiography. (B) The radioactivity of the bands was quantified from three independent experiments as in (A). The amounts of RNA export (% in cytoplasm) were calculated by taking the U6 RNA leakage into account. The averages and the standard deviations are shown. P < 0.01; *P < 0.001; NS, not significant. (C) HeLa cells were transfected with siRNAs as indicated and western blotting was performed with antibodies directed against PHAX, PSF, p54nrb or GAPDH. (D) and (E) HeLa cells were transfected with siRNAs as in (C), and qRT-PCR was performed to quantify the endogenous pre-U1 or pre-U4 atac snRNAs. The averages and the standard deviations from three experiments are shown. (F) HeLa cells were transfected first with siRNAs as indicated and 48 h later with the plasmid expressing U1ΔSm RNA. After 3 h, RNA was extracted from the nuclear and cytoplasmic fractions of the transfected HeLa cells and examined by qRT-PCR for the nucleo-cytoplasmic distribution of the expressed U1ΔSm RNA or endogenous GAPDH mRNA.
	Figure 5. p54nrb and PSF do not accompany U snRNAs to the cytoplasm. (A) Three hundred fmol/oocyte of either m7G-capped or A-capped U1ΔSm RNA was injected into the nucleus of Xenopus oocytes. Twenty hours after injection, the injected oocytes were fractionated and the protein was analyzed by western blotting using the antibodies indicated. Uninjected oocytes were used as a control. (B) HeLa cells were transfected with either the vector (control) or the plasmid expressing U1ΔSm RNA (U1ΔSm) together with a GFP-expressing plasmid (pCDNA3-GFP) to mark transfected cells, and after 24 h, endogenous p54nrb, PSF and PHAX were localized by immunofluorescence staining in GFP-positive cells. DAPI was used to visualize DNA. (C) and (D) Quantitative analyses of the localization of PHAX, p54nrb and PSF with U1ΔSm RNA overexpression in the similar experiments as in (B). The percentage of cells with immunofluorescence signals exclusively in the nucleus (N) or in both the nucleus and cytoplasm (N + C) were calculated. One hundred GFP-positive cells were examined.

References [+] :

Bond, Paraspeckles: nuclear bodies built on long noncoding RNA. 2009, Pubmed