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. 2018 Apr 20;46(7):3774-3790.
doi: 10.1093/nar/gky070.

The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation

Adriana Roithová  1 , Klára Klimešová  1 , Josef Pánek  2 , Cindy L Will  3 , Reinhard Lührmann  3 , David Stanek  1 , Cyrille Girard  3
Affiliations

The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation

Adriana Roithová et al. Nucleic Acids Res. .

Abstract

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

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Figures

Figure 1.
Figure 1.
Sm and SMN binding sites are necessary to target the microinjected U2 snRNA to Cajal bodies. (A–H) In vitro transcribed WT U2 snRNA or deletion mutants thereof were microinjected into the cytoplasm or into the nucleus of HeLa cells. U2 snRNA was labeled with UTP-Alexa-488 (green), coilin, a marker of CBs, was immunolabeled by Alexa-647 (red). Dextran-TRITC 70 kDa (yellow) was used to monitor nuclear or cytoplasmic injection, DNA was stained by DAPI (blue). Small red box in U2 snRNA scheme represents the Sm site. The intensity of the RNA signal in CBs versus the nucleoplasm was determined for each CB, and the average and SEM are shown in graphs next to the micrographs (number of microinjected cells is indicated in the bar). Number of CBs in microinjected cells was counted and plotted. Dotted line indicates number of CBs in control non-injected cells. The scale bar represents 10 μm.
Figure 2.
Figure 2.
The Sm site is necessary for Cajal body targeting of U1, U4 and U5 snRNAs. (A–F) WT or ΔSm U1, U4 and U5 snRNAs were transcribed in vitro and microinjected into the cytoplasm or the nucleus of HeLa cells (green). CBs are marked by coilin immunolabeling (red). Legend is the same as in Figure 1. Small red boxes in snRNA schemes represent the canonical Sm site, the orange box depicts the non-canonical U1 Sm site. (G, H) U2ΔSLI+Sm and U2 snRNA with U1-like Sm site were injected into the nucleus and the cytoplasm of Hela cells. Legend is the same as in Figure 1.
Figure 3.
Figure 3.
Sm and SMN sites are sufficient to target non-coding RNAs into Cajal bodies. (A–D) In vitro transcribed 7SK RNAs (WT or chimeras containing Sm, SMN or both sites) were microinjected into the nucleus or cytoplasm of HeLa cells. Legend is the same as in Figure 1. (E, F) WT Alu or Alu+Sm+SMN sites RNAs were microinjected into the nucleus or cytoplasm of HeLa cells. Legend is the same as in Figure 1. (G, H) WT E. coli SRP RNA or SRP+Sm+SMN sites RNA were microinjected into the nucleus or cytoplasm of HeLa cells. Legend is the same as in Figure 1.
Figure 4.
Figure 4.
Transiently-expressed, truncated snRNAs containing Sm and SMN binding sites accumulate in Cajal bodies. (A) Immunoprecipitation of U2-MS2 and the deletion mutant U2ΔSLI+IIa,b-MS2. RNAs were immunoprecipitated via MS2-YFP by anti-GFP antibodies and detected by silver staining. Co-precipitated proteins were analyzed by Western blotting. With the full-length U2-MS2 RNA we detected all tested proteins (SmB/B′, SF3A3, SNRPB2 and SF3B4), while only SmB/B′ and SNRPB2 co-precipitated with the U2ΔSLI+IIa,b-MS2 RNA. (B) Immunoprecipitation of U4-MS2 and the deletion mutant U4Δ1–64-MS2. Legend as in (A). With the full-length U4-MS2 RNA we detected both tested proteins (SmB/B′ and PRPF31), while only SmB/B′ co-precipitated with U4Δ1–64-MS2 RNA. (C) Hela cells were co-transfected with U2 or U4 snRNAs containing the MS2 loop (green stem loop) and MS2-YFP (green). Coilin was used as a marker of CBs (red). Small red box in snRNA schemes represents the canonical Sm site and blue boxes represent endogenous U2 or U4 promoters. DNA was stained by DAPI. The scale bar represents 10 μm.
Figure 5.
Figure 5.
SmB/B′ but not the SMN protein is essential for Cajal body targeting of snRNA. (A) Depletion of the SmB/B′ protein disrupts targeting of microinjected snRNAs into the Cajal body. SmB/B′ protein was depleted by RNAi and Alexa-488 labeled WT U2 snRNA (green) subsequently microinjected into HeLa cells. Dextran-TRITC 70 kDa was used as a marker of microinjection (yellow). DNA was stained by DAPI (blue). (B, C) SMN protein was depleted by siRNA and either Alexa-488 labeled WT U2 snRNA (B) or core U2 snRNP (C) were microinjected into HeLa cells. Depletion of the SMN protein disrupts CBs, which can be rescued by cytoplasmic microinjection of core U2 snRNP (red). Coilin was used as a marker of CB (green). (C) Dotted lines mark the nucleus of microinjected cells; dashed lines mark the nucleus of non-microinjected cells. The scale bar represents 10 μm.
Figure 6.
Figure 6.
C-terminal tails of SmB, D1 and D3 are important for Cajal body localization. Cells were transfected with plasmids expressing SmB, D1 or D3 protein variants tagged with GFP. Coilin was used as a marker of CBs (red). DNA was stained with DAPI. The scale bar represents 10 μm. Intensity of GFP signal in CBs vs. the nucleoplasm was determined by high-content microscopy. Values normalized to the WT proteins are shown in the table next to the micrographs and non-normalized values are shown in graphs. The average of three experiments and SEM are shown. Statistical significance was assayed by the two tailed t-test and data with a P value < 0.1 are marked by * and P < 0.001 by ***.
Figure 7.
Figure 7.
Partially-assembled snRNP particles induce formation of CBs. (A) Purified 12S U2 snRNP and in vitro reconstituted 15S and 17S U2 snRNPs were analyzed by SDS-PAGE and proteins were visualized by Coomassie staining. (B) TGS1 was knocked down by siRNA and cells were microinjected into the cytoplasm with native 12S U2 snRNP (top panel), in vitro reconstituted 15S U2 snRNP (middle panel) or mature 17S U2 snRNP (bottom panel). FITC-Dextran served as microinjection marker (green) and coilin was visualized by immunostaining (red). (C, D) TGS1 was knocked down by siRNA and cells were microinjected into the cytoplasm with digoxygenin-labeled in vitro-reconstituted U1 snRNP (C) or a native Cyan3-labelled mature U1 snRNP (D) and examined by immunofluorescence 2 h post microinjection using the anti-coilin antibody (C, D) and anti-digoxygenin antibodies (C). Dotted lines mark the nucleus of microinjected cells; dashed lines mark the nucleus of non-microinjected cells. (E) HeLa cells were microinjected in the nucleus with FITC-Dextran (middle panel) or FITC-Dextran together with an equimolar mix of purified SF3a + SF3b complexes (right panel). Left panel shows control non-injected cell. The U2 snRNA was stained by FISH (red) and coilin by immunostaining (green). Insets show a magnified picture of CBs indicated by arrows. Scale bars: 10 μm.
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