mutans, which is one of the principle causative agents of dental caries, has to be elucidated. MicroRNAs (miRNAs) are small noncoding RNAs that are c. 22 nt long. They are found in various species of plants, animals and viruses (Bushati & Cohen, 2007), and normally act as regulators in every major cellular event through inhibitory

mechanisms (He & Hannon, 2004). It has long been known that bacteria contain noncoding small RNAs (sRNAs) that have regulatory functions, other than miRNAs (Gottesman, 2005; Waters & Storz, 2009). sRNAs are usually between 50 and 200 nt in length and have been predicted by computational searches in a variety of bacterial species (Livny & Waldor, 2007). Like miRNAs, sRNAs usually act as post-transcriptional regulators by interacting with the target mRNAs through a variety of mechanisms, including changes in RNA conformation and modulation of the stability

of the specific targets (Waters & Storz, Stem Cell Compound Library isocitrate dehydrogenase inhibitor 2009). Smaller RNAs that have size similar to miRNAs are not well understood in bacteria, although many of them may be found among sequence reads registered in the transcriptome of Escherichia coli (Dornenburg et al., 2010). Streptococcus mutans is the major causative agent of human dental caries and is considered to be the most cariogenic of all of the oral streptococci (Ajdić et al., 2002). The disease occurs when ecologically driven changes in oral biofilms are perturbed and S. mutans is mainly responsible for the formation of the oral biofilms (Burne, 1998). The genome of S. mutans has been fully sequenced and contains c. 2 Mb and 1963 ORFs (Ajdić et al., 2002). This study examined the existence of small (c. 26 nt) RNAs in S. mutans that we subsequently isolated. For this purpose, a deep sequencing (next-generation sequencing) approach was used and more

than 19 million sRNA clones were read. To differentiate these very sRNAs from the bacterial sRNAs (50–250 nt) and well-studied eukaryotic miRNAs, we suggest the term ‘miRNA-size, next small RNA’ (msRNA). Their origin and putative functional significance are discussed. Streptococcus mutans (ATCC 25175) were inoculated into brain heart infusion broth (three independent cultures) and total RNA was extracted from the cultured S. mutans after pooling using the miRNeasy Mini kit (Qiagen, CA) according to the manufacturer’s protocol. RNA was processed and used for deep sequencing by LC Sciences (Houston, TX). An sRNA library was generated from the S. mutans RNA according to Illumina’s sample preparation instructions for Illumina Genome Analyzer IIx (Ilumina Inc., San Diego, CA). The following gives a brief summary of the procedures performed. The total RNA sample was size-fractionated on a 15% Tris–borate–EDTA (TBE)–urea polyacrylamide gel. The RNA fragments of c. 15–50 nt in length were eluted and ethanol-precipitated.