Figure 1.
Domain structure of human Y RNA.
The most stable secondary structure of hY1 RNA is shown as determined by the Mfold v3.2 RNA algorithm. The four conserved key structural elements, including specific protein binding sites, are indicated.
Figure 2.
Human Y RNAs are present in several distinct RNP complexes in cytosolic extract.
The indicated proteins were immunoprecipitated (IP) from HeLa cytosolic extract and associated proteins and RNAs were analysed by Western blotting and qRT-PCR, respectively. (A) Protein analysis of Ro60 and La IPs. Apparent molecular weights of the precipitated protein bands are shown, the asterisk indicates the IgG heavy chain. As a reference, 10% of the input cell extract was loaded on the gel. Where indicated, extract was treated with RNase A prior to IP (RNase). (B) Protein analysis of nucleolin (NCL) IPs. (C) RNA content analysis of the Ro60, La and nucleolin (NCL) IPs. The relative amounts of all four hY RNAs and 5S rRNA in the indicated immunoprecipitates relative to mock immunoprecipitates were determined by qRT-PCR. Mean values of two independent experiments are shown.
Figure 3.
The [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs are not required for the reconstitution of chromosomal DNA replication in vitro.
Immunodepletion of hY RNPs from cytosolic HeLa cell extracts. (A) Depletion of [Ro60/La/hY RNA] RNPs with La-specific antibodies, and (B) depletion of [nucleolin/hY RNA] RNPs with nucleolin-specific antibodies. Ro60, La and nucleolin (NCL) were analysed in the depleted extracts by Western blot analysis. Ponceau stains are shown as loading controls. For mock depletions, unspecific mouse IgG2a antibodies were used alongside purified mouse monoclonal anti-La antibodies, while pre-immune rabbit serum was used alongside the anti-nucleolin rabbit serum. (C) Functional reconstitution of chromosomal DNA replication in the immunodepleted cytosolic extracts. Late G1 phase template nuclei from mimosine-arrested human EJ30 cells were incubated in the indicated depleted extracts. Replication buffer and non-depleted cytosolic extract (cyt) served as negative and positive controls, respectively. Mock M IgG and R IgG indicate unspecific mouse and rabbit antibodies, as in panels A and B. Proportions of replicating nuclei were determined by immunoflorescence microscopy. Mean values of two independent experiments are shown.
Figure 4.
Co-depletion of [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs does not inhibit chromosomal DNA replication in vitro.
[Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs were co-depleted with La- and nucleolin-specific antibodies. (A) Protein levels of nucleolin (NCL), Ro60 and La in the depleted extracts were analysed by Western blotting. Mouse IgG2a antibodies and pre-immune rabbit serum were used together for the mock depletion. (B) Analysis of hY RNA and 5S rRNA levels remaining in the depleted extract. Proportions of the indicated RNA amounts remaining in the extract after [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNP co-depletion were determined by qRT-PCR. Data are shown as percentages of the mock depletion, after normalisation to HPRT mRNA. (C) Functional reconstitution of chromosomal DNA replication in the co-depleted extract. Percentages of replicating nuclei were determined as described for Fig. 3C. Mean values of two independent experiments are shown in panels B and C.
Figure 5.
Ro60 and La proteins do not inhibit chromosomal DNA replication in vitro.
(A) Protein analysis of purified Ro60 by SDS-PAGE and silver staining is shown on the left panel and Western blot analysis on the right panel. Positions of the molecular weight markers (kDa) are indicated. (B) Protein analysis of purified La by SDS-PAGE and Coomassie staining is shown on the left panel and Western blot analysis on the right panel. (C) Formation of [Ro60/La/hY RNA] RNPs from purified components in vitro. 32P-labelled hY1 RNA was incubated with purified Ro60 and/or La at the indicated molar ratios in the presence of a 10-fold molar excess of competitor tRNA. Free RNA was separated from RNPs on native 8% polyacrylamide gels, and radiolabelled RNA was visualised by autoradiography. Positions of free and protein-complexed hY1 RNA are indicated. (D) Functional reconstitution of chromosomal DNA replication in the presence of excess recombinant Ro60 and/or La. Template nuclei were incubated in limiting cytosolic extract (40 µg cyt) with recombinant proteins as indicated. Replication buffer and non-limiting cytosolic extract (cyt) were used as negative and positive controls, respectively. Proportions of replicating nuclei were determined as described for Fig. 3C. Mean values and standard deviations of n = 15–18 independent experiments are shown.