Abstract
MicroRNAs (miRNAs) are known to play important roles in various cellular processes and stress responses. MiRNAs can be identified by analyzing reads from high-throughput deep sequencing. The reads realigned to miRNA precursors besides canonical miRNAs were initially considered as sequencing noise and ignored from further analysis. Here we reported a small-RNA species of phased and half-phased miRNA-like RNAs different from canonical miRNAs from cassava miRNA precursors detected under four distinct chilling conditions. They can form abundant multiple small RNAs arranged along precursors in a tandem and phased or half-phased fashion. Some of these miRNA-like RNAs were experimentally confirmed by re-amplification and re-sequencing, and have a similar qRT-PCR detection ratio as their cognate canonical miRNAs. The target genes of those phased and half-phased miRNA-like RNAs function in process of cell growth metabolism and play roles in protein kinase. Half-phased miR171d.3 was confirmed to have cleavage activities on its target gene P-glycoprotein 11, a broad substrate efflux pump across cellular membranes, which is thought to provide protection for tropical cassava during sharp temperature decease. Our results showed that the RNAs from miRNA precursors are miRNA-like small RNAs that are viable negative gene regulators and may have potential functions in cassava chilling responses.
Highlights
MicroRNAs form a class of endogenous, 20–22nt long regulatory RNA molecules
We fully utilized the small-RNA deep sequencing profiling data that we collected from cassava that were subjected to four cold treatments[27]
It has been shown that many Arabidopsis miRNA precursors can yield multiple distinct ~21-nt miRNAs in phasing along the same precursors[15], many of which are conserved in other plants, including rice, Poplus, Medicago and Moss[15]
Summary
MicroRNAs (miRNAs) form a class of endogenous, 20–22nt long regulatory RNA molecules. With the growing popularity of Generation sequencing of small RNAs, mapping of sequencing reads to genome sequences has often resulted in many non-negligible small RNA reads beyond miRNAs and miRNA*s, which can align perfectly to the “stem” regions of pre-miRNA precursors. Some of these small RNA species even present in high www.nature.com/scientificreports/. Bologna et al.[21] and Schapire et al.[22] systemically analyze miRNA processing intermediates during the biogenesis of small RNAs in Arabidopsis, by using an approach called Specific Parallel Amplification of RNA Ends (SPARE) They found that at least 4 different processing pathways are involved in the generation of most miRNAs in Arabidopsis. The above method has revealed an unexpected complexity of the miRNA processing pathways acting upon both ancient and evolutionarily young sequences, and shown that members of the same miRNA family can be produced in different ways
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