Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo

Abstract

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B'' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.

Figure 5.

Effects of SF3a depletion on nuclear structures and transcription. (A) HeLa cells were transfected with siRNAs 60/2 or 120/2, fixed 60 h posttransfection, and immunostained with pAb60 or pAb120 and Texas Red-anti-rabbit, and anti-SC35 and FITC-anti-mouse as indicated. The right panels show computer-generated overlays of the images. Arrows indicate cells depleted of SF3a. (B) and (C) HeLa cells transfected with siRNA 60/2 were fixed 60 h posttransfection and immunostained with pAb60 and FITC-anti-rabbit, and anti-p80-coilin (B) or anti-SMN (C) and Texas Red-anti-mouse as indicated. Nuclei were visualized by Hoechst staining (left panels). (D) HeLa cells transfected with siRNA 60/2 were pulse-labeled 60 h posttransfection with FU for 45 min. Incorporation of FU into newly synthesized RNA was detected by confocal microscopy after staining with anti-BrdU and FITC-anti-mouse (right). SF3a60 was visualized with pAb60 and Texas Red anti-rabbit (middle). Nuclei were visualized by Hoechst staining (left). Scale bars, 10 μm.

Figure 6.

Depletion of U2 snRNA and U2-B″ from speckles, but not from CBs after knockdown of SF360. (A) HeLa cells transfected with siRNA 60/2 were fixed 60 h posttransfection. Cells were stained with pAb60 and Texas Red-anti-rabbit, and anti-U2-B″ and FITC-anti-mouse as indicated. The right panel shows a computer-generated overlay of the left and middle images. (B) Cells transfected and fixed as in A were incubated with pAb60 and AMCA-anti-rabbit and hybridized with FAM- and Cy3-conjugated oligonucleotides complementary to U2 and U4 snRNAs, respectively, as indicated. (C) and (D) Confocal images of cells transfected with siRNA 60/2 and stained for U2-B″ and U2 snRNA as in panels A and B 60 h posttransfection. Arrows mark SF3a60-depleted cells; arrowheads indicate CBs. Scale bars, 10 μm.

Figure 1.

Effects of SF3a and SF1 depletion on viability of HeLa cells. HeLa cells were transfected with siRNAs 60/2, 66/1, 120/2, SF1/1, and GL2, or mock-transfected in the absence of siRNA. Cell viability was monitored 24, 48, and 72 h posttransfection. The average of three independent experiments (except for GL2) is shown.

Figure 3.

Effects of SF3a and SF1 depletion on protein expression. (A) HeLa cells were transfected with siRNAs indicated above the figure or oligofectamine alone (-). A triple transfection (Σ) was performed with siRNAs 60/2, 66/1, and 120/2. Total cell lysates were prepared 72 h posttransfection, separated by 7.5% SDS-PAGE, and transferred to nitrocellulose. Membranes were incubated with the antibodies indicated on the left. The bottom panel shows a representative Ponceau S–stained membrane as a loading control. (B) Total lysates of cells transfected with siRNA SF1/1 or oligofectamine alone (-) were prepared 48 h posttransfection and analyzed as in A. (C) HeLa cells transfected with siRNAs 60/2, 66/1, 120/2, SF1/1, or oligofectamine alone (-) were metabolically labeled with [³⁵S]methionine and ³⁵S-cysteine. Cell lysates were separated by 10% SDS-PAGE, and translation products were visualized by autoradiography. (D) Cytoplasmic RNA was isolated from HeLa cells transfected with siRNAs 60/2, 66/1, 120/2, or oligofectamine alone (-) and translated in vitro. A control reaction was performed in the absence of RNA (no RNA). Translation products were separated by 12% SDS-PAGE and visualized by autoradiography.

Figure 2.

Effects of SF3a and SF1 depletion on pre-mRNA accumulation. (A) HeLa cells were transfected with siRNAs 60/2, 66/1, 120/2, all three (Σ) or oligofectamine alone (-). Total RNA isolated 60 h posttransfection was used for RT-PCR with primers specific for endogenous SF3a60, SF3a66, SF3a120, SF1 and histone H3F1 RNAs, and RNA transcribed from the transiently transfected β-globin reporter plasmid pAdCMV-glob. (B) HeLa cells were transfected with siRNAs SF1/2, 60/2 or oligofectamine alone (-). RNA isolation and RT-PCR was performed as in A. Reaction products were separated in agarose gels. RT-PCR products derived from pre-mRNAs and mRNAs are marked by asterisks and arrowheads, respectively. The sizes of DNA markers (M) are shown on the left.

Figure 4.

Effects of SF3a depletion on U2-B″, SmB/B′, p80-coilin, and SC35 expression. (A) Cell lysates prepared from HeLa cells transfected with siRNAs 60/2, 66/1, 120/2, or oligofectamine alone (-) were prepared 60 h later and separated by 12% SDS-PAGE followed by detection of the proteins indicated on the left by Western blotting. The bottom panel shows the Ponceau S–stained membrane as a loading control. (B) HeLa cells were transfected with siRNAs 60/2, 66/1, 120/2, or oligofectamine alone (-). Total RNA isolated 60 h posttransfection was used for RT-PCR with primers specific for SC35 and p80-coilin RNAs. Reaction products were analyzed as in Figure 2.

Figure 7.

Analysis of U2 snRNPs and snRNAs in SF3a60-depleted cells. (A) HeLa cells were mock-treated or transfected with siRNA 60/2. Aliquots of small-scale nuclear extracts (normalized for protein concentration) prepared 72 h posttransfection from mock-treated (lane 2) and siRNA-transfected (lane 3) cells were incubated with a labeled oligoribonucleotide complementary to the 5′ end of U2 snRNA. Partially purified 17S and 15S U2 snRNPs (lane 1) were used as markers for the migration of U2 particles. The extract from siRNA-treated cells was supplemented with the U2 snRNP fraction (lane 4). SnRNP complexes were separated by native 4% PAGE and visualized by autoradiography. The migration of U2 complexes is indicated on the left. (B) SnRNAs present in total RNA isolated from cells 60 h after transfection with siRNAs 60/2, 66/1, and 120/2 or oligofectamine alone (-) were analyzed by Northern blotting. The snRNAs are indicated on the right.