Co-depletion of [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs does not inhibit chromosomal DNA replication in vitro.

<p>[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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013673#pone-0013673-g003" target="_blank">Fig. 3C</a>. Mean values of two independent experiments are shown in panels B and C.</p

The [Ro60/La/hY RNA] and [nucleolin/hY RNA] RNPs are not required for the reconstitution of chromosomal DNA replication in vitro.

<p>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.</p

Domain structure of human Y RNA.

<p>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.</p

Human Y RNAs are present in several distinct RNP complexes in cytosolic extract.

<p>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.</p

Nucleotide Contributions to the Structural Integrity and DNA Replication Initiation Activity of Noncoding Y RNA

Noncoding Y RNAs are small stem–loop RNAs that are involved in different cellular processes, including the regulation of DNA replication. An evolutionarily conserved small domain in the upper stem of vertebrate Y RNAs has an essential function for the initiation of chromosomal DNA replication. Here we provide a structure–function analysis of this essential RNA domain under physiological conditions. Solution state nuclear magnetic resonance and far-ultraviolet circular dichroism spectroscopy show that the upper stem domain of human Y1 RNA adopts a locally destabilized A-form helical structure involving eight Watson–Crick base pairs. Within this helix, two G:C base pairs are highly stable even at elevated temperatures and therefore may serve as clamps to maintain the local structure of the helix. These two stable G:C base pairs frame three unstable base pairs, which are located centrally between them. Systematic substitution mutagenesis results in a disruption of the ordered A-form helical structure and in the loss of DNA replication initiation activity, establishing a positive correlation between folding stability and function. Our data thus provide a structural basis for the evolutionary conservation of key nucleotides in this RNA domain that are essential for the functionality of noncoding Y RNAs during the initiation of DNA replication