The multifunctional protein p54nrb/PSF recruits the exonuclease XRN2 to facilitate pre-mRNA 3' processing and transcription termination

Abstract

Termination of RNA polymerase II transcription frequently requires a poly(A) signal and cleavage/polyadenylation factors. Recent work has shown that degradation of the downstream cleaved RNA by the exonuclease XRN2 promotes termination, but how XRN2 functions with 3'-processing factors to elicit termination remains unclear. Here we show that XRN2 physically associates with 3'-processing factors and accumulates at the 3' end of a transcribed gene. In vitro 3'-processing assays show that XRN2 is necessary to degrade the downstream RNA, but is not required for 3' cleavage. Significantly, degradation of the 3'-cleaved RNA was stimulated when coupled to cleavage. Unexpectedly, while investigating how XRN2 is recruited to the 3'-processing machinery, we found that XRN2 associates with p54nrb/NonO(p54)-protein-associated splicing factor (PSF), multifunctional proteins involved in several nuclear processes. Strikingly, p54 is also required for degradation of the 3'-cleaved RNA in vitro. p54 is present along the length of genes, and small interfering RNA (siRNA)-mediated knockdown leads to defects in XRN2 recruitment and termination. Together, our data indicate that p54nrb/PSF functions in recruitment of XRN2 to facilitate pre-mRNA 3' processing and transcription termination.

Figure 1.

XRN2 is associated with 3′-processing factors. (A) Structure of human XRN2. 5′–3′ exonuclease domains are shown by black boxes. (B) HeLa NE was immunoprecipitated with anti-CstF-64 antibody. (Lane 3) IPs were resolved and blotted with anti-XRN-2 antibody. (Lane 2) Control IP was performed with anti-CstF-64 antibody plus blocking peptide. (Lane 1) Ten percent of NE was loaded to indicate the position of XRN2. (C) Reciprocal IPs with anti-XRN2 antibody show association with CstF-64. NE was immunoprecipitated with anti-XRN2 antibody. (Lane 3) IPs were resolved and visualized by immunoblot with CstF-64 antibody. (Lane 2) XRN2 antibody was omitted in the mock IP. (Lane 1) NE (12.5%) was loaded to indicate the position of CstF-64. (D) Schematic representation of the β-actin locus and the locations of the PCR primers used for ChIP analysis. Each exon is indicated by a black box. The transcription initiation site is indicated by an arrow. (E) XRN2 accumulates after the poly(A) site. ChIP analysis of XRN2 on the β-actin locus is shown. The Y-axis shows the signal-to-noise ratio (S/N) of XRN2 IP relative to control IP (anti-XRN2 antibody plus blocking peptide). Error bars show the standard deviation from three independent replicates.

Figure 2.

Immunodepletion of XRN2 results in accumulation of 3′ downstream RNA. (A) Anti-XRN2 antibody prevents degradation of 3′ downstream RNA. In vitro cleavage assays were performed with NE and adenovirus L3 (Ad-L3) pre-mRNA. (Lane 2) Anti-XRN2 antibody was added to the reaction mixture. Ad-L3 pre-mRNA, 5′-cleaved RNA, and 3′ downstream RNA are indicated by arrows. DNA size markers are shown on the left side. (B) In vitro cleavage assay of Apolipoprotein CI (ApoCI) pre-mRNA.(Lane 2) Cleavage reaction with anti-XRN2 antibody was performed. (C) Immunodepletion of XRN2. (Lane 2) XRN2-depleted NE (ΔXRN2) was resolved by SDS-PAGE and blotted with the indicated antibodies. Immunoblots of depleted (lane 3) and untreated (lane 1) NE are shown. (D) Cleavage reactions of Ad-L3 (lanes 1–3) and ApoCI (lanes 4–6) pre-mRNAs were performed with XRN2-depleted (lanes 2,5), mock-depleted (lanes 3,6), and untreated (lanes 1,4) NE.

Figure 3.

Degradation of 3′ downstream RNA is coupled with 3′ processing. (A) Preparation of RNA substrates for degradation assays. The 3′-cleaved RNA (lane 3) was gel purified from cleaved Ad-L3 pre-mRNA (lane 1). (B, lanes 1–4) Time-course analysis of in vitro cleavage reaction was performed with NE and Ad-L3 pre-mRNA. (Lanes 5–8) The identical reaction was performed with 3′ downstream RNA instead of Ad-L3 pre-mRNA. The 3′ downstream RNA was prepared by gel purification of cleaved Ad-L3 pre-mRNA. (C) Degradation of 3′ downstream RNA was quantified at a time interval between 5 ∼ 10 min and 10 ∼ 15 min of the reactions. (D) Quantitation of XRN2 protein in NE. Indicated amounts of NE (lane 1) and recombinant, C-terminal-truncated XRN2 (lanes 2–4) were resolved and visualized by immunoblot with XRN2 antibody. (E) XRN2 ribonuclease reactions were performed under typical ribonuclease conditions (lanes 1–4) or 3′ cleavage conditions (lanes 5–8) with 3′ downstream RNA and increasing amounts (0, 10, 20, and 50 ng) of XRN2.

Figure 4.

The C-terminal region (786–950) of XRN2 is necessary and sufficient for protein–protein interactions. (A) Schematic representation of Flag-tagged wild-type and XRN2 truncations. After transient transfection of XRN2 expression vectors into HeLa cells, cell lysates were used as probes for Far Western assay. (B) NE was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with either wild-type XRN2 (lane 2) or XRN2 truncations (XRN2 Δ2–Δ6, lanes 3–7). (Lane 1) A lysate of mock-transfected HeLa cells is used as a negative control. (C) Immunoblots of XRN2-truncated proteins. Proteins were detected by anti-Flag antibody.

Figure 5.

PSF–p54nrb is associated with XRN2. (A) Affinity purification of glutathione S-transferase GST-XRN2Δ6-bound proteins. (Lane 1) Bacterially expressed GST-XRN2Δ6 was incubated with NE. (Lane 2) After extensive washing, GST-XRN2Δ6-bound proteins were eluted with glutathione and developed by SDS-PAGE. (B) Far Western analysis shows that the IP of p54 contains XRN2-interacting proteins. Immunoprecipitated NEs with anti-p54nrb (lane 2), preimmune serum (lane 5), and anti-actin (lane 6) were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with Flag-tagged XRN2Δ6. (C) NEs were immunoprecipitated with anti-p54 (lane 3) and preimmune serum (lane 2). IPs were resolved and visualized by Western blot with anti-XRN2 antibody. (Lane 1) Ten percent of NE was loaded to indicate the position of XRN2 protein. (D) Reciprocal IPs with anti-XRN2 revealed interaction with p54/PSF. NEs were immunoprecipitated with anti-XRN2 (lane 3) and preimmune serum (lane 2). IPs were resolved and blotted with anti-PSF antibody. (Lane 1) Ten percent of NE was loaded to indicate the position of PSF. (E) p54 localizes throughout an active gene. ChIP analysis of p54 on the β-actin locus is shown (see Fig. 1D). The Y-axis shows the signal-to-noise ratio (S/N) of p54 IP relative to control IP (p54 antibody plus blocking peptide).

Figure 6.

Immunodepletion of p54/PSF results in accumulation of 3′-cleaved RNA. (A) Immunodepletion of p54/PSF. (Lane 1) p54/PSF-depleted NE (Δp54–PSF) was resolved by SDS-PAGE and blotted with the indicated antibodies. (Lane 2) Mock depletion (Mock) is also shown. (B) Cleavage reactions of Ad-L3 pre-mRNA were performed with p54–PSF-depleted (lane 1) and mock-depleted (lane 2) NE.

Figure 7.

Knockdown of p54 leads to defective termination and XRN2 recruitment. (A) siRNA knockdown of p54. Whole-cell lysates of HeLa cells transfected with p54 (lane 2) and control (lane 1) siRNA were resolved by SDS-PAGE and blotted with the indicated antibodies. (B) ChIP analysis of Pol II on the β-actin locus is shown (see Fig. 1D). The Y-axis shows the signal-to-noise ratio (S/N) of Pol II IP relative to control IP (Pol II antibody plus blocking peptide). (C) p54 is required for recruitment of XRN2 to Pol II EC. ChIP analysis of XRN2 was performed as described in Figure 1E. (D) The XRN2 ChIP signals are presented normalized to the Pol II ChIP signals with or without p54 depletion.