Seq and CLIP through the miRNA world

Abstract

High-throughput sequencing of RNAs crosslinked to Argonaute proteins reveals not only a multitude of atypical miRNA binding sites but also of miRNA targets with atypical functions, and can be used to infer quantitative models of miRNA-target interaction strength.

Figure 1

High-throughput methods for sequencing small RNAs and their targets. Conceptual protocols highlighting the differences between the methods for deep sequencing of (a) small RNAs and of (b-e) small RNA targets (PAR-CLIP (b), CLASH (c), HITS-CLIP (d), iCLIP (e)). Ni-NTA, nickel nitrilotriacetic acid; Gu-HCL, guanidine hydrochloride; PNK, polynucleotide kinase.

Figure 2

The multi-faceted miRNA biogenesis and miRNA interaction with targets. miRNAs are processed mainly by Drosha-DGCR8 in the canonical pathway, but also by the lariate-debranching enzyme in the nucleus, and by Dicer (from other non-coding RNAs such as tRNAs and snoRNAs) and Ago2 in the cytoplasm. Although miRISC generally regulates the stability and translation rate of target mRNAs, other long RNAs feed back on the miRNA regulation by sequestering miRNAs from their direct targets.