Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells

Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication.HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.One major posttranscriptional regulatory pathway, RNA interference (RNAi), is mediated by small noncoding RNAs (sncRNAs) [1]. Over recent years, the importance of the diverse classes of sncRNAs has been widely recognized and their impact on various biological processes demonstrated across a broad variety of organisms [2]. The most intensively studied class of sncRNAs are the 20-25 nucleotides long microRNAs (miRNAs) which play a crucial role in posttranscriptional regulation of gene expression [3].Despite technological advances sncRNAs of low abundance have remained difficult to identify. To date, the most frequently employed method to derive sncRNAs is the generation of cDNA libraries encoding sncRNAs by, rather rate limiting, cloning and sequencing procedures [4]. While this technique allows the identification of sncRNAs of medium to high frequency with notable success, it rem