Abstract
BackgroundRo ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.Methodology/Principal FindingsWe first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.Conclusions/SignificanceWe conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.
Highlights
Ro ribonucleoprotein particles (Ro RNPs) are soluble complexes in vertebrate cells which are detected by autoimmune antibodies from patients suffering from systemic lupus erythematosis or Sjogren’s syndrome [1,2]
Results hY RNAs are present in several distinct RNP complexes in human cytosolic extract To confirm that Ro60, La and nucleolin stably interact with hY RNAs, we first immunoprecipitated these proteins from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these immunoprecipitates (Fig. 2)
As it was unknown if these Y RNA binding proteins play a role in DNA replication, we investigated their function in this process
Summary
Ro ribonucleoprotein particles (Ro RNPs) are soluble complexes in vertebrate cells which are detected by autoimmune antibodies from patients suffering from systemic lupus erythematosis or Sjogren’s syndrome [1,2]. Depletion of hY RNAs from human cell extracts inhibits the initiation step of chromosomal DNA replication in a mammalian cell-free system [7,9] This inhibition can be overcome by the addition of pure Y RNAs. Recently, we have shown that an evolutionarily conserved double-stranded RNA motif present in the upper stem of vertebrate Y RNAs is essential and sufficient for their function in DNA replication in mammalian cells [10]. We have shown that an evolutionarily conserved double-stranded RNA motif present in the upper stem of vertebrate Y RNAs is essential and sufficient for their function in DNA replication in mammalian cells [10] These data indicate that Y RNAs are functionally active, but it is unknown if any of the associated Ro RNP proteins play a role in DNA replication. We investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication
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