Fam118B, a newly identified component of Cajal bodies, is required for Cajal body formation, snRNP biogenesis and cell viability

Abstract

Cajal bodies are specialized and dynamic compartments in the nucleus that are involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Because of the dynamic and varied roles of Cajal bodies, it is of great interest to identify the components of Cajal bodies to better understand their functions. We performed a genome-wide screen to identify proteins that colocalize with coilin, the marker protein of Cajal bodies. In this study, we identified and characterized Fam118B as a newly discovered component of Cajal bodies. Fam118B is widely expressed in a variety of cell lines derived from various origins. Overexpression of Fam118B changes the canonical morphology of Cajal bodies, whereas depletion of Fam118B disrupts the localization of components of Cajal bodies, including coilin, the survival of motor neuron protein (SMN) and the Sm protein D1 (SmD1, also known as SNRPD1). Moreover, depletion of Fam118B reduces splicing capacity and inhibits cell proliferation. In addition, Fam118B associates with coilin and SMN proteins. Fam118B depletion reduces symmetric dimethylarginine modification of SmD1, which in turn diminishes the binding of SMN to this Sm protein. Taken together, these data indicate that Fam118B, by regulating SmD1 symmetric dimethylarginine modification, plays an important role in Cajal body formation, snRNP biogenesis and cell viability.

Keywords: Cajal body; Coilin; Fam118B; SMN; Symmetric dimethylarginine.

Fig. 1.

Fam118B is a newly identified component of Cajal bodies. (A) The specificity of the Fam118B polyclonal antibody was confirmed by western blot analysis. HeLa cells were subjected to two rounds of treatment with the indicated siRNA and the cell lysates were analyzed by western blot analysis with the anti-Fam118B antibody. Asterisks indicate non-specific bands; sictrl, control siRNA. (B) Fam118B is expressed in different cancer cell lines. Cell lysates from HepG2, HeLa, U2OS, HCT116, 293T and MDA-MB-231 cells were collected. Western blot analysis was performed to detect the protein levels of endogenous Fam118B and coilin. β-actin is included as a loading control. (C) Fam118B concentrates in Cajal bodies. HeLa cells were treated with Triton X-100 or left untreated, and were subjected to immunofluorescent staining using the indicated antibodies recognizing Fam118B (green) or coilin (red). Scale bar: 10 µm. (D) Fam118B colocalizes with coilin in various cancer cell lines. The indicated cancer cell lines were pre-extracted with Triton X-100 and stained with antibodies against Fam118B (green) and coilin (red). Scale bar: 5 µm. In C and D, the nuclei were stained with DAPI (blue).

Fig. 2.

Fam118B associates with the major components of Cajal bodies. (A) Fam118B interacts with coilin and SMN. HeLa lysates were immunoprecipitated (IP) by using polyclonal anti-Fam118B or anti-coilin antibodies, and were analyzed by western blot analysis with the indicated antibodies. (B) Immunoprecipitation using control IgG or anti-SMN antibody was performed in extracts prepared from HeLa cells. The presence of coilin or Fam118B in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (C) A GST pull-down assay was performed using immobilized control GST or GST–coilin fusion protein on agarose beads followed by incubation with bacterially purified MBP-tagged Fam118B protein. The in vitro interaction of Fam118B with coilin was assessed by immunoblotting with the anti-MBP antibody. The arrow shows the migrating band of GST–coilin. (D) The formation of Fam118B foci is coilin dependent. HeLa cells depleted of coilin (siCoilin) were immunostained with polyclonal anti-Fam118B (green) and anti-coilin (red). Scale bar: 5 µm.

Fig. 3.

Amino acid residues 190–232 of Fam118B are required for targeting to Cajal bodies. (A) Schematic representations of the functional domain of Fam118B based on CDD and the mutant constructs used in this study. FL, full length. (B) The expression of wild-type (FL) and mutant Fam118B proteins was examined by western blot analysis with the anti-FLAG antibody. (C) HeLa cells were transiently transfected with constructs encoding the indicated Fam118B wild-type or mutant proteins and were stained with anti-FLAG (green) and anti-coilin (red) antibodies. Arrows indicate the foci in which FLAG-tagged Fam118B colocalizes with coilin. (D) M1, M4 and M6 mutants localize in the nucleoli. HeLa cells were transiently transfected with constructs encoding the M1, M4 or M6 mutants of Fam118, fixed and stained with anti-FLAG (red) and anti-fibrillarin (green) antibodies. In C and D, the nucleus was stained with DAPI (blue). Scale bars: 5 µm. (E) The percentage of Fam118B wild-type or mutant protein that colocalized with coilin is presented. The data show the mean±s.d. of three independent experiments; 50 cells per experiment.

Fig. 4.

Depletion of Fam118B causes the disassembly of Cajal bodies. (A) The depletion of Fam118B did not affect the protein level of coilin or SMN, but resulted in a reduction in SmD1 protein levels. HeLa cells depleted of Fam118B (siFam118B) were analyzed by western blotting with the indicated antibodies. (B) The depletion of Fam118B disrupted canonical Cajal body formation. HeLa cells transfected with control siRNA (siCtrl) or Fam118B siRNA were immunostained with polyclonal anti-Fam118B (green) and anti-coilin (red) antibodies. (C) The percentage of cells with canonical coilin foci is shown. (D) The depletion of Fam118B disrupted SMN foci. Cells were immunostained with anti-Fam118B (green) and anti-SMN (red) antibodies. (E) The percentage of cells with SMN foci in the nucleus is shown. (F) The depletion of Fam118B caused SmD1 to be redistributed to the nucleolus. Cells were immunostained with anti-Fam118B (green) and anti-SmD1 (red) antibodies (upper panel), or with anti-fibrillarin (green) and anti-SmD1 (red) antibodies (lower panel). (G) The percentage of cells with SmD1 in the nucleolus is shown. In B, D and F, the nucleus was stained with DAPI (blue). Scale bars: 5 µm. In C, E and G, the data show the mean±s.d. of three independent experiments; 50 cells per experiment.

Fig. 5.

Depletion of Fam118B causes a reduction in splicing capacity and cell growth. (A) HeLa cells or HeLa cells stably expressing RNAi-resistant wild-type Fam118B (+WT) were subjected to two rounds of treatment with Fam118B (siFam118B) or coilin (siCoilin) siRNA, followed by western blot analysis using the indicated antibodies. Asterisks indicate non-specific bands. siCtrl, control siRNA. (B) A schematic representation of the artificial splicing reporter. The locations of the primers and the expected sizes of PCR products are shown. FP, forward primer; RP, reverse primer. (C) The depletion of Fam118B influenced the splicing of the artificial reporter. HeLa cells or HeLa cells stably expressing RNAi-resistant wild-type Fam118B were subjected to two rounds of treatment with Fam118B or coilin siRNA, followed by pSI transfection. The isolated RNA was subjected to RT-PCR to amplify the pSI message by using the indicated primers. (D) The ratio of unspliced to spliced PCR products normalized to the internal control was compared between the samples. (E) The abundance of spliced mRNA for eight endogenous genes was quantified by qRT-PCR, using primers spanning a particular splice junction. Levels of splicing efficiency are given relative to control (mock siRNA-treated cells, siCtrl) and are normalized to the levels of NEAT1 RNA, which served as an internal control. (F) The depletion of Fam118B resulted in a reduction in cell proliferation. HeLa cells were seeded and subjected to transfection with Fam118B siRNA or coilin siRNA on the first day after seeding. Fam118B+WT cells were transiently infected with retroviral supernatant to restore Fam118B expression on the second day, and then were counted for the following 4 days. In D–F, the data show the mean±s.d. of three independent experiments.

Fig. 6.

Depletion of Fam118B decreases the affinity of SmD1 for SMN and reduces the amount of sDMA modification of SmD1. (A) HeLa cells that stably expressed an SmD1 fusion protein containing the HA-FLAG tag were subjected to transfection with control (siCtrl) or Fam118B (siFam118B) siRNA. Ectopically expressed SmD1 protein was immunoprecipitated by using M2 beads and was detected by using anti-FLAG antibody. The amount of co-precipitated SMN was detected by western blotting using the anti-SMN antibody. The sDMA modification of SmD1 was detected by using the anti-SYM10 antibody. (B) Quantification of the amount of SMN protein that was co-immunoprecipitated with ectopic SmD1 (normalized to control) in control or Fam118B-depleted cells. (C) HeLa cells were subjected to transfection with control or Fam118B siRNA and were harvested. Western blot analysis was performed with indicated antibodies. (D) Quantification of the amount of sDMA-modified endogenous SmD1 (normalized to control) in control or Fam118B-depleted cells. In B and D, the data show the mean±s.d. of three independent experiments.