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.

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.

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.