Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA  Yingqun Huang, Joan A. Steitz  Molecular Cell  Volume 7, Issue 4, Pages 899-905 (April 2001) DOI: 10.1016/S1097-2765(01)00233-7

Figure 1 Schematic Diagrams of Constructs (A) A 22-nt export element from the mouse histone H2a mRNA. The hatched rectangle represents the open reading frame (ORF) of the H2a gene numbered relative to the histone transcription start site with the 101-nt sequence indicated. The 22-nt element is shown with the five nucleotide substitutions needed to produce an inactive element. The putative 9G8 and SRp20 binding sites are indicated by the bold and the thin lines above the sequence, respectively. (B) Constructs used for transfection into COS 7 cells. The large open rectangles represent the human or Xenopus β-globin cDNA sequence. The arrows and the solid ovals indicate the direction of transcription and the globin polyadenylation signals, respectively. An example of cryptic splicing from the human globin coding region to the vector sequence downstream of the globin poly(A) site is indicated. The small solid rectangle, with the 22-nt element marked by an open box, represents the 101-nt histone nuclear export sequence inserted at the unique Nco I site. Deletion of the 22-nt element is indicated by a thin line, whereas small open boxes represent single, double, and quadruple repeats of the 22-nt element inserted at the Nco I site. The quadruple small gray boxes represent four repeated copies of the mutant 22-nt element inserted at the Nco I site. In H-XβG, the hatched rectangle represents a mouse histone H2a sequence (H2a-2 to 494 nt relative to the H2a transcription start site). RNAs expressed from H-XβG are efficiently polyadenylated and partition between the nucleus and cytoplasm of transfected cells in a reproducible manner, thus serving as an internal control for nucleocytoplasmic fractionation (Huang and Carmichael, 1997). Diagrammed below the constructs are the riboprobes (thin lines) used in RNase protection assays, with the protected fragments (thick lines) indicated. The tilted line in the 3′ end probe the represents vector sequence. (C) Constructs (not drawn to scale) used for microinjection as well as for in vitro UV cross-linking: The four tandem open or gray boxes represent four repeated copies of the wild-type or the mutant 22-nt element, respectively, with the horizontal thin line indicating vector sequences. The DraI and NcoI restriction sites were used to linearize the plasmids before transcription to produce 4XSV and m4XSV RNA for microinjection and 4X and m4X RNA for UV cross-linking experiments, respectively. Numbers are in nucleotides Molecular Cell 2001 7, 899-905DOI: (10.1016/S1097-2765(01)00233-7)

Figure 2 The 22-nt Element Promotes the Nuclear Export of Intronless mRNAs (A–C) Autoradiograms of RNase protection assays of RNAs expressed in transfected cells. The human β-globin plasmids in Figure 1B were transfected individually into COS 7 cells along with the internal control plasmid H-XβG. Forty-eight hr later, nuclear and cytoplasmic RNAs were prepared and subjected to RNase protection assays using a mixture of the 3′ end and the control probes. c = cytoplasmic; n = nuclear; Control = polyadenylated RNA expressed from H-XβG; RT = read-through globin or globin-related RNA; Cleaved = globin or globin-related RNA whose 3′-end has been correctly polyadenylated; c/n ratio = cytoplasmic versus nuclear ratio of the cleaved globin or globin-like RNAs normalized to the internal control RNA. (D) Results of a Xenopus oocyte microinjection experiment. A mixture of three radioactive RNAs, U1, U6, and one reporter RNA (4XSV or m4XSV), was injected into Xenopus oocyte nuclei. RNAs were extracted 2 hr later, and two oocyte equivalents of RNA were loaded on an 8% denaturing polyacrylamide gel. Reporter RNA = 5′-capped RNA transcribed from 4XSV or m4XSV; U1 = 5′-capped U1 RNA transcribed from pT7U1; U6 = 5′-uncapped U6 RNA transcribed from pT7U6; Reporter c/n ratio = c/n ratio of the reporter RNA; U1 c/n ratio = c/n ratio of U1 RNA. The experiment was repeated three times, and similar results were obtained Molecular Cell 2001 7, 899-905DOI: (10.1016/S1097-2765(01)00233-7)

Figure 3 Antibodies Identify 9G8 and SRp20 as Important Factors for Export (A) The 22-nt element is specifically recognized by SRp20 and 9G8. Radioactively labeled mutant (m4X) or wild-type (4X) RNA (see Figure 1C) was incubated with HeLa nuclear extract followed by cross-linking. After treatment with RNase A, proteins were separated by 15% SDS-PAGE. Lanes 1 and 2 show total UV cross-linking patterns, whereas lanes 3 through 8 show subsequent immunoprecipitation of cross-linked proteins by anti-SRp20 (αSRp20, lanes 3 and 4), anti-9G8 (α9G8, lanes 5 and 6), or anti-SF2/ASF (αSF2/ASF, lanes 7 and 8). The reaction volumes in lanes 3–6 were double and in lanes 7 and 8 were quadruple those in lanes 1 and 2. Shown on the left are the mobilities of protein size markers in kDa. The cross-linked SRp20 and 9G8 bands in lane 2 are marked by stars. (B) Antibodies to SRp20 and 9G8 block the nuclear export of an element-containing RNA. A mixture of three radioactive RNAs (4XSV, U1, and U6), together with one of the indicated antibodies (5 mg/ml), was injected into the nuclei of Xenopus oocytes. Two hours later, eight oocytes with blue nuclei were collected for each group, and nuclei were fractionated from cytoplasm. Two oocyte equivalents of RNA isolated from each fraction were resolved on an 8% denaturing polyacrylamide gel. The c/n ratios of 4XSV and U1 RNAs are displayed at the bottom of the gel. (C) Antibodies interfere with RNA binding by SRp20 and 9G8. The indicated antibody, or antibodies, or PBS, was incubated with 32P-CTP labeled 4X RNA and HeLa nuclear extract followed by UV cross-linking. After RNase A treatment, the cross-linked products were resolved by 15% SDS-PAGE, with SRp20 and 9G8 indicated by the stars. Protein size markers in kDa are shown on the left Molecular Cell 2001 7, 899-905DOI: (10.1016/S1097-2765(01)00233-7)

Figure 4 SRp20 and 9G8 Associate with Polyadenylated RNAs in Both Cellular Compartments In Vivo Growing monolayer HeLa cells were exposed (lanes 3 and 4) or not (lanes 1 and 2) to UV light, followed by purification of nuclear and cytoplasmic poly(A)+ RNAs with covalently bound proteins on oligo d(T) columns. Eluates from the columns were treated with nucleases, and bound proteins were analyzed by Western blotting using the indicated antibodies. “−,” no UV exposure; “+,” with UV exposure; c, cytoplasmic fraction; n, nuclear fraction. These experiments were repeated two times, with similar patterns observed Molecular Cell 2001 7, 899-905DOI: (10.1016/S1097-2765(01)00233-7)