Abstract
BackgroundThe various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs.ResultsEight 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.ConclusionsHIV-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.
Highlights
The various classes of small noncoding RNAs are important regulators of gene expression across divergent types of organisms
Enrichment and selection of low abundant HIV-1 sncRNAs by hybridization capture One aim of our study was to derive an effective selection strategy for low abundant sncRNAs which would allow 1) to determine the presence or absence of sncRNAs in a given setting and 2) to allow the characterization of the full spectrum of sncRNAs generated by HIV-1 where conflicting reports have been published which suggested that either no or only extremely low numbers of HIV-1 sncRNAs are evolved in infected cells
As outlined in the following procedures, we achieved this by introducing a specific selection step which enriched for HIV-1 derived sequences
Summary
The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. 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 remains less effective in defining low abundant sncRNAs. Alternate approaches have employed microarray- and PCR-based technologies to detect and quantify sncRNAs [4,5]. High-throughput sequencing techniques have been applied [6,7,8]
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