6 research outputs found
Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans.
Stem bulge RNAs (sbRNAs) are a family of small non-coding stem-loop RNAs present in Caenorhabditis elegans and other nematodes, the function of which is unknown. Here, we report the first functional characterisation of nematode sbRNAs. We demonstrate that sbRNAs from a range of nematode species are able to reconstitute the initiation of chromosomal DNA replication in the presence of replication proteins in vitro, and that conserved nucleotide sequence motifs are essential for this function. By functionally inactivating sbRNAs with antisense morpholino oligonucleotides, we show that sbRNAs are required for S phase progression, early embryonic development and the viability of C. elegans in vivo. Thus, we demonstrate a new and essential role for sbRNAs during the early development of C. elegans. sbRNAs show limited nucleotide sequence similarity to vertebrate Y RNAs, which are also essential for the initiation of DNA replication. Our results therefore establish that the essential function of small non-coding stem-loop RNAs during DNA replication extends beyond vertebrates.This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC doctoral training grant DTG (BB/F016581/1) and grant BB/K013378/1).This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/jcs.166744
Structural and functional analysis of four non-coding Y RNAs from Chinese hamster cells: identification, molecular dynamics simulations and DNA replication initiation assays
Abstract
Background
The genes coding for Y RNAs are evolutionarily conserved in vertebrates. These non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate cells. However thus far, no information is available about Y RNAs in Chinese hamster cells, which have already been used to detect replication origins and alternative DNA structures around these sites. Here, we report the gene sequences and predicted structural characteristics of the Chinese hamster Y RNAs, and analyze their ability to support the initiation of chromosomal DNA replication in vitro.
Results
We identified DNA sequences in the Chinese hamster genome of four Y RNAs (chY1, chY3, chY4 and chY5) with upstream promoter sequences, which are homologous to the four main types of vertebrate Y RNAs. The chY1, chY3 and chY5 genes were highly conserved with their vertebrate counterparts, whilst the chY4 gene showed a relatively high degree of diversification from the other vertebrate Y4 genes. Molecular dynamics simulations suggest that chY4 RNA is structurally stable despite its evolutionarily divergent predicted stem structure. Of the four Y RNA genes present in the hamster genome, we found that only the chY1 and chY3 RNA were strongly expressed in the Chinese hamster GMA32 cell line, while expression of the chY4 and chY5 RNA genes was five orders of magnitude lower, suggesting that they may in fact not be expressed. We synthesized all four chY RNAs and showed that any of these four could support the initiation of DNA replication in an established human cell-free system.
Conclusions
These data therefore establish that non-coding chY RNAs are stable structures and can substitute for human Y RNAs in a reconstituted cell-free DNA replication initiation system. The pattern of Y RNA expression and functionality is consistent with Y RNAs of other rodents, including mouse and rat
Functional roles of non-coding Y RNAs.
Non-coding RNAs are involved in a multitude of cellular processes but the biochemical function of many small non-coding RNAs remains unclear. The family of small non-coding Y RNAs is conserved in vertebrates and related RNAs are present in some prokaryotic species. Y RNAs are also homologous to the newly identified family of non-coding stem-bulge RNAs (sbRNAs) in nematodes, for which potential physiological functions are only now emerging. Y RNAs are essential for the initiation of chromosomal DNA replication in vertebrates and, when bound to the Ro60 protein, they are involved in RNA stability and cellular responses to stress in several eukaryotic and prokaryotic species. Additionally, short fragments of Y RNAs have recently been identified as abundant components in the blood and tissues of humans and other mammals, with potential diagnostic value. While the number of functional roles of Y RNAs is growing, it is becoming increasingly clear that the conserved structural domains of Y RNAs are essential for distinct cellular functions. Here, we review the biochemical functions associated with these structural RNA domains, as well as the functional conservation of Y RNAs in different species. The existing biochemical and structural evidence supports a domain model for these small non-coding RNAs that has direct implications for the modular evolution of functional non-coding RNAs.Research in the authors’ laboratory has been funded by grants from Cancer Research UK (CRUK), the Association for International Cancer Research (AICR) and the Biotechnology and Biological Sciences Research Council (BBSRC)
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