Figure 1.
RBPL-1 is conserved in C. elegans. a.
Predicted structure of RBPL-1 (TAG-214). The conserved domains DWNN, zinc knuckle, and RING finger are indicated by blue, red, and gray boxes, respectively. The nuclear localization signal (NLS) is indicated by a green box. b. Protein sequence alignment highlights the conservation of the DWNN, Zn knuckle, and RING finger domains among human, rat, and C. elegans RBBP6 proteins. c. Full-length rbpl-1 mRNA cloned by RT-PCR. d. RBPL-1 expressed in E. coli. Upper: SDS-PAGE analysis of the bacterially expressed GST-RBPL-1 fusion protein, indicated by the red arrow. Lower: western blotting analysis of GST-RBPL-1 fusion protein (the red arrow) using an anti-GST antibody.
Figure 2.
RNAi for rbpl-1 reduces oocyte number. a.
RNAi knockdown of rbpl-1 results in reduced embryo number. The earlier RNAi is applied, the more severe the defects. The empty vector L4440 was used as an RNAi control. The grow temperature was 15°C. (YA = Young Adult, A = Adult). b. rbpl-1 RNAi silenced hermaphrodites crossed with him-5 males produce no more progeny than control RNAi hermaphrodites only. c. Adult rbpl-1 RNAi worms show abnormal germline development. There is no difference between the rbpl-1 RNAi and control RNAi in germline development at L3 stage. In adults, the oocytes are abnormal and reduced in rbpl-1 worms. Some oocytes in rbpl-1 RNAi adult possess multiple sets of chromosomes (shown in the inset) (H2B::GFP fluorescence indicates the nucleus of germ cells). Scale bar: 30 µm. d. rbpl-1 RNAi in either soma or germline significantly reduces egg production. In the strain NL2098, RNAi is effective in the germline and intestine, while in the strain CB4856, RNAi is effective only in the soma. *P<0.05.
Figure 3.
RNAi for rbpl-1 causes defects of germ cell proliferation. a.
The posterior end of the germline showing the mitotic and transition zones. Nuclei are labeled by H2B::GFP. Scale bar: 10 µm. b. Fewer mitotic cells are observed in rbpl-1 RNAi worms than in control worms by immunofluorescence. The arrow indicates mitotic cells labeled with anti-P-H3S10. Scale bar: 10 µm. c. The mitotic index in rbpl-1 RNAi worms is much lower than in control worms. Mitotic index: the number of cells in mitotic stage (staining by anti-P-H3S10) divided by the total cell number in mitotic zone (staining by DAPI).
Figure 4.
RNAi for rbpl-1 results in defects of intestine development a.
The intestine (indicated by dashed line) has nearly disappeared in the rbpl-1 worms compared with controls. Scale bar: 25 µm. b. Continuous RNAi of rbpl-1 in the intestine leads to a progressively reduced brood size through generations. *P<0.05. c. The production of vitellogenin (indicated by vit-2::GFP) is inhibited by RNAi silencing of rbpl-1 (arrow head indicates the oocyte). Scale bar: 50 µm. d. Fluorescence imaging showing that lipid content is largely reduced upon silencing of rbpl-1 in the whole worm (N2) or intestine only (VP303), but not in the hypodermis only (NR222). Scale bar: 25 µm. e. Survival time is shortened when silencing rbpl-1 in the whole worm (N2) or only in the intestine (VP303), whereas RNAi for rbpl-1 in the hypodermis (NR222) does not affect life span. f. Quantification of fluorescence intensity in Figure 4D indicates that the lipid content decreases significantly when silencing rbpl-1 in the whole worm (N2), or only in the intestine (VP303), but not when restricted to the hypodermis (NR222). *P<0.01.
Figure 5.
RBPL-1 is expressed in diverse tissues and localized to the nucleolus. a.
RBPL-1::GFP is expressed in multiple tissues, including neurons, intestine, spermatheca, and vulva. Scale bar: 20 µm. b. RBPL-1::GFP is localized to the nucleolus of intestine cells. The bottom left image indicates the nucleolar localization of RBPL-1 within an intestine nucleus (the dashed line indicates the nucleus). Scale bar: 50 µm. c. Dynamics of RBPL-1 during embryogenesis. A transient disappearance of RBPL-1 fluorescence occurs from metaphase to telophase. Membrane and RBPL-1 are marked by GFP, and nuclei are shown by H2B::mCherry. Scale bar: 2 µm.
Table 1.
List of polyadenylation factors predicted to interact with RBPL-1 according to the interactome on WormBase.