A subclass of small RNAs is encoded by exons of protein-coding genes

Small RNAs regulate gene expression in species across the tree of life. miRNAs, which impact a variety of cellular and physiological processes ranging from development and stress adaptation to host defense, are one of the best characterized classes of small RNA. Many miRNAs are produced from longer non-coding transcripts generated from host genes via a series of RNA cleavage reactions. The location of a small RNA within a host gene can shape the processing of the mature small RNA. For example, a type of miRNAs derived from host gene intronic sequence, referred to as miRtrons, are Drosha-independent and reliant on splicing for biogenesis. Relatedly, processing of a small RNA from an exon of a protein-coding mRNA, in principle, may destabilize it and compromise translation of the host gene. Prior to extensive transcriptome analysis, informatics analyses identified six human miRNAs embedded in exons of protein-coding genes and experimental studies have characterized additional anecdotal examples. Still, whether protein-coding mRNAs encoding small RNAs represent an appreciable class of host genes given the now recognized complexity of the transcriptome is unclear.

Our analysis finds 201 small RNAs (118 human and 83 mouse) encoded by expressed exons of protein-coding genes (5'-UTR, CDS, 3'-UTR). Forty-six of these cases (29 human and 17 mouse) are also present in MirGeneDB which includes the most up-to-date miRNA classifications. Many of these small RNAs are poorly characterized with 96% of the protein-coding host gene relationships identified here not previously known. Furthermore, the identification of nearly fifty human and mouse small RNAs embedded within coding exons of canonical ORFs suggests that overlapping hybrid genes might be more common than previously appreciated in higher organisms. Expression analysis for a subset of these small RNAs indicates that many display differential expression across human tissues with the pattern correlating significantly with the expression of the candidate protein-coding host gene.

Overall, our analysis suggests that the number of protein-coding transcripts serving as host genes is greater than previously recognized. Our small RNA host gene classifications may serve as a resource to shed new light on small RNA biology, specific host genes, and gene regulation.