A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Abstract

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.

Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA

“…The 83-nt primary hY5 transcript, which is reported to form a very stable hairpin structure (van Gelder et al 1994;Maraia et al 1996) and thus likely renders the 23-and 31-nt regions inaccessible, also triggers substantially lower cell deaths. Interestingly, Gardiner et al (2009) and Wang et al (2014) reported a double-stranded version of the critical 8 nt found in the RNY5 sRNA (5 ′ GUAGUGGG3 ′ ) to be sufficient for RNY1 to support the initiation of DNA replication. However, in our studies it is clear that the hY5 31-and 23-nt processed products triggered cell death is attenuated in the presence of their complementary strands.…”

“…However, in our studies it is clear that the hY5 31-and 23-nt processed products triggered cell death is attenuated in the presence of their complementary strands. Although Gardiner et al (2009) did not report if a single-stranded version of this sequence was capable of supporting the initiation of replication, one possibility is that the single-stranded 31-and 23-nt sRNAs cause inappropriate and perhaps uncontrolled DNA replication signals in primary cells, triggering cell death. Such processed RNY5-stimulated signals might be less effective in cancer cell lines given their characteristic loss of DNA replication controls inherent with transformed cells.…”

“…3F). Gardiner et al (2009) reported that a conserved doublestranded sequence motif in the upper stem of all vertebrate Y-RNAs correlated with their participation in initiating DNA replication. Each of the products processed from the 5 ′ side of RNY5 in vivo and in vitro contains a single-stranded version of this motif.…”

“…Since these original observations, multiple descriptions of other ribonucleoprotein complexes involving Y RNAs have been described, prompting the hypothesis that Y-RNAs may have multiple functions based on the protein partners present in the complexes (Sim et al 2009). More recently, support for this hypothesis has been provided by reports that cellular Y-RNAs have specific functional roles including forming part of the initiation of DNA replication complex (Christov et al 2006;Gardiner et al 2009), the chaperoning of misfolded RNAs (Chen et al 2003;Gardiner et al 2009), and assisting in the quality control of 5S ribosomal RNAs (Hogg and Collins 2007). Correlated with each of these functional roles has been the identification of a variety of distinct proteins associated with the Y-RNAs involved.…”

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

“…In extracts from human somatic cells, reconstitution of chromosomal DNA replication in isolated cell nuclei requires a class of non-coding RNAs termed Y RNAs (Christov et al, 2006;Gardiner et al, 2009;Krude et al, 2009). These are small, structured stem-loop RNAs of between 69-112 nucleotides in length, which are conserved in vertebrate evolution Farris et al, 1995;Mosig et al, 2007;Perreault et al, 2007).…”