Small RNA molecules such as miRNAs, piRNAs and siRNAs have become recognised as vital in the regulation of many important processes such as the fine-tuning of gene expression during development, and repression of transposons. Here we present the first in-depth characterisation of small RNAs present in the reproductive tissues of the honeybee. We have performed small RNA sequencing on ovaries, spermatheca, sperm, fertilised and unfertilised eggs and testes.
364 novel miRNAs were detected, with the majority coming from the testes and spermatheca samples. The expression pattern of known and novel miRNAs differs dramatically between tissue types with semen in particular having a highly divergent miRNA expression pattern.
piRNAs are a class of small RNA between 24-31 nt long with a strong 5’U bias and 2’O-methylation at the 3’ end. They are generally void of sequence conservation and the ability to form hairpin structures from flanking genomic DNA, and thus differ greatly from miRNAs. They are usually transcribed from both large, repeat-rich, uni- and bi-directional clusters distributed throughout the genome. Their primary function differs based on tissue type, and can be either suppression of transposon activity or regulation of endogenous gene expression. Using custom scripts we have identified a total of 168 piRNA clusters. The clusters appear to be non-randomly distributed throughout the genome. The average cluster size is 10kb, and the average length of sequences located within the clusters is 28bp.
Finally, the semen samples are composed of approximately 60% tRNA fragments (tRFs). tRFs are a highly conserved, ancient type of small RNA molecule that have been suggested to have a role in the transmission of epigenetic signals between generations in mice. Surprisingly, the tRFs found in honeybee semen are almost identical to those implicated in epigenetic inheritance in mouse semen. The implications of this finding will be discussed.