Functional microRNAs reside within the RNA induced silencing complex (RISC), bound to an Argonaute protein. They control gene expression by guiding RISC to target mRNAs causing RNA degradation or translational repression. We have applied Argonaute HITS-CLIP (High-Throughput Sequencing of Cross-linking Immunoprecipitation) [1] to assess the relative function of microRNAs, transfected microRNA mimics and other small RNAs of diverse genomic origin which have historically been dismissed as RNA degradation products.
We show that Argonaute binding of small RNAs processed from RNA stem-loop structures is not limited to miRNAs but is widely observed for a variety of constitutively expressed RNAs including small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs), vault RNAs (vRNAs), small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and some pseudogenes with no previously known function. We interrogate the functionality of non-canonical miRNAs using miRNA sensor reporter assays to reveal several novel regulatory RNAs. More widely however, this work highlights that only abundant microRNAs have detectable functionality and the vast majority of putative non-canonical miRNAs are very lowly abundant. We give evidence against postulations that several highly abundant small RNAs from tRNAs, vRNAs and yRNAs are miRNAs, but we give novel insight to biosynthesis and processing of these non-coding RNAs with elusive functions. Using degradome-seq, a sequencing approach able to identify endonucleolytic RNA cleavage [2], we note endonuclease processing of RNA stem-loop structures on a genome-wide scale.
Finally, we have assessed the functionality of transfected siRNAs commonly used as miRNA mimics, revealing that the majority of transfected RNA is not functionally bound to Argonaute but rather reside non-functionally within lysosomes and associated vesicles. Despite this, qRT-PCR measurement of total cellular RNA is commonly used to assess miRNA mimic functionality misleadingly suggesting supraphysiological miRNA abundance. Equally, transfected miRNA inhibitors also reside within vesicles and qRT-PCR measurements from cell lysates does not necessarily demonstrate their efficacy [3].