Sumoylation modulates the assembly and activity of the pre-mRNA 3' processing complex

Abstract

Eukaryotic pre-mRNA 3'-end formation is catalyzed by a complex set of factors that must be intricately regulated. In this study, we have discovered a novel role for the small ubiquitin-like modifier SUMO in the regulation of mammalian 3'-end processing. We identified symplekin, a factor involved in complex assembly, and CPSF-73, an endonuclease, as SUMO modification substrates. The major sites of sumoylation in symplekin and CPSF-73 were determined and found to be highly conserved across species. A sumoylation-deficient mutant was defective in rescuing cell viability in symplekin small interfering RNA (siRNA)-treated cells, supporting the importance of this modification in symplekin function. We also analyzed the involvement of sumoylation in 3'-end processing by altering the sumoylation status of nuclear extracts. This was done by the addition of a SUMO protease, which we show interacts with both symplekin and CPSF-73, or by siRNA-mediated depletion of ubc9, the SUMO E2-conjugating enzyme. Both treatments resulted in a marked inhibition of processing. The assembly of a functional polyadenylation complex was also impaired by the SUMO protease. Our identification of two key polyadenylation factors as SUMO targets and of the role of SUMO in enhancing the assembly and activity of the 3'-end-processing complex together reveal an important function for SUMO in the processing of mRNA precursors.

FIG. 1.

Symplekin is modified by SUMO-2/3 in HeLa cells. (A) HeLa cells were cotransfected with a plasmid encoding Flag-tagged symplekin along with empty vector (lane 1) or vectors encoding HA-SUMO-1, -2 or -3 (lanes 2, 3, and 4). Cell lysates were subjected to Western blot analysis with anti-Flag or antiactin antibodies. Higher-molecular-weight forms and the unmodified form of symplekin are indicated by closed and open arrowheads, respectively. (B) 293 cells were cotransfected with a plasmid encoding His-tagged SUMO-3 (lanes 1 and 4) or with Flag-tagged symplekin plus empty vector (lanes 2 and 5) or vectors encoding His-tagged SUMO-3 (lanes 3 and 6). Lysates were prepared under denaturing conditions and subjected to Ni-NTA chromatography. Inputs (lanes 1, 2, and 3) and the Ni-NTA-bound proteins (lanes 4, 5, and 6) were analyzed by Western blotting with anti-Flag antibodies. The unmodified (open arrowhead) and SUMO-modified (bracket) forms of symplekin are indicated. (C) Symplekin immunoglobulin G or control immunoglobulin G was used to immunoprecipitate proteins from HeLa NE, and IPs were analyzed by Western blotting with antisymplekin (left panel), anti-SUMO-2/3 (middle panel), and anti-SUMO-1 (right panel) antibodies. The closed arrowheads indicate the slower-migrating form of symplekin. Positions of molecular weight markers are indicated. WB, Western blot; +, present; −, absent.

FIG. 2.

CPSF-73 is modified by SUMO-2/3. (A) HeLa cells were transfected with a plasmid encoding Flag-tagged CPSF-73 along with empty vector (lane 1) or plasmids encoding HA-SUMO-1, -2 or -3 (lanes 2, 3, and 4). Whole-cell lysates were subjected to Western blotting with anti-Flag and antiactin antibodies. (B) HeLa cells were transfected with a plasmid encoding Flag-tagged CPSF-73 along with empty vector (lanes 1 and 3) or a plasmid encoding His-tagged SUMO-3 (lanes 2 and 4), and cell lysates were partially purified under denaturing conditions by Ni-NTA chromatography as described in Materials and Methods. Inputs (lanes 3 and 4) and bound fractions (lanes 1 and 2) were subjected to Western blotting with anti-Flag antibodies. Higher-molecular-weight forms of CPSF-73 are indicated by closed arrowheads, and unmodified CPSF-73 is indicated by an open arrowhead. (C) Antibodies to CPSF-73 or control immunoglobulin Gs were used to immunoprecipitate proteins from HeLa NEs, and IPs were analyzed by Western blotting with anti-CPSF-73 (left panel), anti-SUMO-2/3 (middle panel), and anti-SUMO-1 (right panel) antibodies. Higher-molecular-weight forms of CPSF-73 are indicated by closed arrowheads. Positions of molecular weight markers are indicated. WB, Western blot; +, present; −, absent.

FIG. 3.

The major sites of sumoylation in symplekin and CPSF-73 are conserved among species. (A) A plasmid encoding HA-SUMO-3 was cotransfected with plasmids encoding Flag-tagged wt symplekin or the indicated mutants into 293T cells, and lysates subjected to immunoblotting with anti-Flag antibodies. The sumoylated forms of symplekin are indicated by arrows. The panel below is a CLUSTALW alignment of human, mouse, and Xenopus symplekin. The bottom panel shows an amino acid alignment of human symplekin and yeast Pta1. Conserved lysines are boxed. (B) 293T cells were cotransfected with plasmids encoding His-SUMO-3 and Flag-tagged wt CPSF-73 (lanes 2 and 12) or the indicated mutants. Lysates were prepared and subjected to immunoblotting with anti-Flag antibodies. Sumoylated forms of CPSF-73 are indicated by arrows. The bottom panel shows a CLUSTALW amino acid alignment of human, zebrafish, D. discoideum and S. cerevisiae homologs of CPSF-73. Conserved SUMO acceptor lysines are boxed. WB, Western blot; +, present; −, absent.

FIG. 4.

Cells expressing a sumoylation-deficient mutant of symplekin are inviable. (A) Symplekin siRNA1 or control siRNAs were cotransfected with pcDNA3 empty vector, pcDNA3-wt symplekin, or pcDNA3-mt symplekin with Lipofectamine 2000 in six-well plates as described in Materials and Methods. Adherent cells were harvested 48 h, 72 h, and 96 h after transfection from three independent experiments and counted, and the mean value plotted as a percentage of cells in the control transfection (control siRNA plus empty vector). Standard error bars are indicated. (B) Western blot analysis with antisymplekin antibodies (top panel) and antiactin (bottom panel) of cells harvested at 48 h (lanes 1 through 4) and 72 h (lanes 5 through 8) after transfection. mt, mutant; WB, Western blot; +, present; −, absent.

FIG. 5.

Sumoylation in HeLa NE is downregulated by ubc9 siRNA and SENP2 protease, which interacts specifically with symplekin and CPSF-73. (A) GST-SUMO-3 was added to reaction mixtures containing HeLa NE under polyadenylation conditions. Reaction products were analyzed by Western blotting with anti-GST antibodies. (B) HeLa cells were transfected with control or ubc9 siRNAs (si), and NEs were subjected to Western blotting with anti-ubc9 and antiactin antibodies (left panels). Control siRNA NE, ubc9 siRNA NE, or buffer (right panel, lanes 1 to 3, respectively) was added to the reaction mixtures described above. (C) Reactions were performed as described for panel A in the presence of buffer (lane 2); 50 ng, 200 ng, 500 ng, and 1 μg of WT SENP2 (lanes, 3, 4, 5, and 6); or 1 μg of DM SENP2 (lane 7) and analyzed by Western blotting with GST antibodies. Lane 1 is a negative control without NE. In all panels, free GST-SUMO-3 is indicated by a closed arrow and higher-molecular-weight GST-SUMO-3 conjugates are indicated by an open bracket. (D) HeLa NE was incubated overnight with 8 μg GST or GST-SENP2 proteins bound to glutathione beads. Ten percent of input NE, bound proteins from GST, and GST-SENP2 beads were subjected to Western blot analysis with anti-CPSF-73, antisymplekin, anti-CstF-64, and antiactin antibodies as indicated. WB, Western blot.

FIG. 6.

Knockdown of ubc9 by RNAi results in loss of cleavage and polyadenylation activity. (A) ³²P-labeled pG3SVL-A pre-RNA was incubated under standard cleavage conditions in NEs prepared from control siRNA-treated HeLa cells or ubc9 siRNA-treated cells. RNAs were resolved by denaturing PAGE. The unprocessed pre-RNA (SVL) and the 5′ and 3′ cleavage products are indicated. (B) ³²P-labeled pG3SVL-A precleaved pre-mRNA substrate (pre-SVL) was incubated as described for panel A under polyadenylation conditions, and RNAs were resolved by denaturing PAGE. The pre-mRNA substrate (precleaved SVL) and the polyadenylated products are indicated. (C) NEs made from control siRNA-treated cells (lane 1) or ubc9 siRNA-treated cells (lane 2) were analyzed by Western blotting with the antibodies indicated on the right. (D) HeLa cells were transfected with control siRNA (lane 1) or ubc9 siRNA (lane 2) and harvested, and extracts analyzed by Western blotting with antisymplekin (top panel), anti-ubc9 (middle panel), or antiactin (bottom panel) antibodies. The higher-molecular-weight forms of symplekin and the unmodified form are indicated by a bracket and an arrow, respectively. WB, Western blot.

FIG. 7.

Desumoylation of NE by the SUMO protease SENP2 inhibits 3′ processing and blocks the formation of polyadenylation-specific complexes. (A) ³²P-labeled pG3L3-pre-RNA was incubated under cleavage/polyadenylation conditions following preincubation with buffer (lane 1) or 50 ng, 100 ng, 500 ng, and 1 μg wt SENP2 (lanes 2, 3, 4, and 5, respectively). RNAs were resolved by denaturing PAGE. (B) ³²P-labeled pG3SVL-A pre-RNA (SVL) was incubated under cleavage conditions following preincubation with buffer (lane 1); 50 ng, 100 ng, 500 ng, and 1 μg wt SENP2 (lanes 2, 3, 4, and 5, respectively); or 1 μg DM SENP2 (lane 6). RNAs were resolved by denaturing PAGE. (C) ³²P-labeled pG3SVL-A precleaved pre-mRNA (pre-SVL) was incubated under polyadenylation conditions after preincubation as described above. Pre-RNA, 5′ cleaved products, and the polyadenylated products are indicated on the right in all panels. (D) Standard cleavage/polyadenylation reaction mixtures using ³²P-labeled pG3SVL-A pre-RNA were assembled with buffer (lane 1) or NE (lane 3). Reaction mixtures using ³²P-labeled pG3SVL-AAAAAA mutant RNA were assembled with NE (lane 2). Following incubation, complexes were resolved by nondenaturing PAGE. Positions of free pG3SVL-A pre-RNA substrate polyadenylation-specific complexes and nonspecific complexes are indicated. (E) Standard reaction mixtures were assembled with NE as above, and preincubation was carried out with buffer (lane 1); 50 ng, 100 ng, 500 ng, and 1 μg wt SENP2 (lanes 2, 3, 4, and 5, respectively); or 1 μg DM SENP2 (lane 6) before the addition of ³²P-labeled pG3SVL-A pre-RNA. Complexes were analyzed as described for panel D.