Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies

Abstract

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.

Figure 1.

Validation of the targeting of hTGS1 and the SMN protein by siRNA duplexes. (A) Specific RNAi-mediated depletion of mRNAs encoding hTGS1 or SMN revealed by real-time RT-PCR analysis. HeLa cells were transfected either with luciferase siRNAs (control) or with siRNAs targeting hTGS1 or SMN. After 24 h cells were harvested, total RNA was isolated, and real-time RT-PCRs were performed. The diagram shows the relative levels of the target mRNAs compared with mRNA levels from cells transfected with control siRNAs directed against the nonhuman protein luciferase (set to 100%). (B) Knockdown of hTGS1 and SMN proteins by RNAi. Cells were treated as in A and harvested 72 h after transfection with siRNAs against hTGS1 (ΔhTGS1) or 48 h after transfection with siRNAs against SMN (ΔSMN), and the levels of the corresponding proteins were determined by immunoblotting (α-hTGS1 and α-SMN, respectively). (C) Cell survival after siRNA transfection. HeLa cells were harvested 24, 48, and 72 h after siRNA transfection and counted. The graphs show the growth curve of HeLa cells depleted of hTGS1 or SMN. For comparison, the growth rate of HeLa cells transfected with siRNAs targeting the nonhuman protein luciferase (control) and the essential splicing factor hPrp8 are also shown.

Figure 2.

hTGS1 is required for efficient hypermethylation of U snRNAs in vitro. (A) Extract isolated from HeLa cells depleted of hTGS1 by RNAi shows a reduced hypermethylation efficiency. In vitro–reconstituted radiolabeled U1 snRNP containing an m⁷G cap was incubated with S100 extract from HeLa cells depleted of hTGS1 (ΔhTGS1), SMN (ΔSMN), or the nonhuman protein luciferase (control) by RNAi. The reaction mixture was then split, and the U1 snRNP was immunoprecipitated with the m₃G-cap–specific antibody R1131, which immunoprecipitates only hypermethylated U1 snRNPs (product; top panel), and with the mAb H-20, which recognizes both m⁷G and m₃G caps and thus binds to all U1 snRNPs in the reaction (total U1; bottom panel). (B) The results of three independent hypermethylation assays as described in A are summarized in the graph. The hypermethylation efficiency was calculated by the ratio of hypermethylation products immunoprecipitated with the R1131 antibody to the total U1 snRNA immunoprecipitated with the H-20 antibody. The hypermethylation efficiency was normalized to control extract (set to 1). Error bars, SD. (C) Recombinant hTGS1 restores the hypermethylation activity in hTGS1-depleted extract. Either recombinant hTGS1(aa 577–851) encompassing the methyltransferase domain (WT) or an inactive hTGS1(aa 577–851) mutant carrying a D695A mutation that affects S-AdoMet binding (D695A) was added to S100 extract depleted of hTGS1 (ΔhTGS1). The hypermethylation activity was then assayed as in A. (D) Summary of two independent hypermethylation assays as described in C and quantified as in B.

Figure 3.

Depletion of hTGS1 or SMN leads to a relocalization of coilin into numerous small foci in the nucleoplasm and into nucleoli. (A) HeLa cells transfected with siRNAs targeting luciferase (control), hTGS1 (ΔhTGS1), or SMN (ΔSMN) were examined by indirect immunofluorescence. Cells were stained with antibodies against coilin (a, d, and g) and fibrillarin as a marker for nucleoli (b, e, and h). Panels c, f, and i show the merged picture of the two panels above, where coilin is shown in green and fibrillarin in red. Yellow indicates overlying signals. (B) HeLa cells as described in A were stained with antibodies against Nop58 (a, d, and g) and coilin (b, e, and h). Panels c, f, and i show the merged picture of the two panels above, where Nop58 is shown in green and coilin in red. Yellow indicates overlying signals.

Figure 4.

Sm class U snRNPs are predominantly localized in speckles and the U6 snRNP protein p110 is distributed diffusely throughout the nucleoplasm in cells depleted of hTGS1 or SMN. (A) HeLa cells transfected with siRNAs targeting luciferase (control), hTGS1 (ΔhTGS1), or SMN (ΔSMN) were examined by indirect immunofluorescence. Cells were stained with an autoimmune serum specific for Sm proteins (a, d, and g) and with an antibody against coilin (b, e, and h). Panels c, f, and i show the merged picture of the two panels above, where Sm proteins are shown in green and coilin in red. Yellow indicates overlying signals. (B) HeLa cells as described in A were examined using FISH for U2 snRNA (a, d, and g) and counterstained with an anti-coilin antibody (b, e, and h). Panels c, f, and i show the merged picture of the two panels above, where U2 snRNA is shown in green and coilin in red. Yellow indicates overlying signals. (C) HeLa cells as described in A were stained with antibodies against p110 (a, d, and g) and coilin (b, e, and h). Panels c, f, and i show the merged picture of the two panels above, where p110 is shown in green and coilin in red. Yellow indicates overlying signals.

Figure 5.

Depletion of PHAX leads to a distribution of U snRNPs and snoRNP components comparable to knockdown of hTGS1 or SMN. (A–E) HeLa cells transfected with siRNAs targeting luciferase (control) or PHAX (ΔPHAX) were examined by using indirect immunofluorescence or FISH. Cells were stained with an antibody against fibrillarin (A, a and d), an antibody against Nop58 (B, a and d), an autoimmune serum specific for Sm proteins (C, a and d), an oligonucleotide against U2 snRNA (D, a and d), an antibody against p110 (E, a and d), and an antibody against coilin (A–E, b and e). Panels c and f show the merged picture of the two panels above, where fibrillarin, Nop58, Sm proteins, U2 snRNA, or p110 are shown in green and coilin in red. Yellow indicates overlying signals.

Figure 6.

snoRNP components, but not spliceosomal U snRNPs, form detectable foci in coilin-depleted cells. (A) Specific RNAi-mediated depletion of coilin mRNA revealed by real-time RT-PCR analysis. HeLa cells were transfected with either the luciferase control siRNAs or with siRNAs targeting coilin. After 24 h cells were harvested, total RNA was isolated, and real-time RT-PCRs were performed. The diagram shows the relative level of the coilin mRNA compared with cells transfected with control siRNAs (set to 100%). (B) Knockdown of coilin protein by RNAi (Δcoilin). Cells were treated as in A and harvested 72 h after transfection, and the level of coilin protein analyzed by immunoblotting (α-coilin). (C) Cell survival after siRNA transfection. HeLa cells were harvested 24, 48, and 72 h after siRNA transfection and counted. The graphs show the growth curve of HeLa cells depleted of coilin. For comparison the growth rate of HeLa cells transfected with siRNAs targeting the nonhuman protein luciferase (control) and the essential splicing factor hPrp8 were determined in parallel. (D) HeLa cells transfected with siRNAs targeting luciferase (control) or coilin (Δcoilin) were examined using indirect immunofluorescence or FISH. Cells were stained with antibodies against coilin (a and g), fibrillarin (b and h), or Nop58 (c and i), with an autoimmune serum specific for Sm proteins (d and j), with an oligonucleotide against U2 snRNA (e and k), or with an antibody against p110 (f and l). In panels b–f, CBs are indicated with an arrow.