UC Riverside

Small RNAs are short, non-coding RNA molecules that have been identified in a wide range of species across all three domains of life. In mammals, canonical small RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) are tissue-specifically distributed and can regulate gene expression at both transcriptional and post-transcriptional levels, associating with various fundamental functions such as gene silencing and retrotransposon control. While miRNAs and piRNAs have been extensively investigated, the existence and mechanism of other non-canonical mammalian small RNAs remain underexplored.With the extensive use of high-throughput sequencing technologies in the past decade, small RNA diversity is rapidly growing. However, the conventional small RNA library construction method lacks the detection capability for non-canonical small RNAs that carry specific terminal and internal modifications, especially for tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNAs). In addition, existing downstream bioinformatics tools are mostly focused on analyzing canonical small RNAs such as miRNAs and piRNAs, while the annotation of other small RNAs remains rudimentary or ignored. To reveal a panoramic view of small RNAs, a small RNA annotation pipeline (that is, SPORTS) is developed to simultaneously and comparatively annotate both canonical and non-canonical small RNAs, along with RNA modification prediction capacity. Moreover, a small RNA library preparation procedure (that is, PANDORA-seq) is optimized to comprehensively capture modified small RNAs such as tsRNAs and rsRNAs. The improved RNA-seq and bioinformatics strategy leads to a new and surprising small RNA landscape that tsRNAs and rsRNAs have a higher abundance than canonical small RNAs in a majority of mouse and human tissues/cells that have been examined. Those mammalian tsRNAs and rsRNAs also exhibit tissue- and cell-specific patterns and the expression level of those small RNAs are dynamically altered during the generation of induced pluripotent stem cells (iPSCs) based on PANDORA-seq. Those newly identified small RNAs also display translational regulation during embryonic stem cell differentiation and have a role in regulating embryonic stem cell lineage fate based on the transcriptomic changes after their transfection.