MicroRNAs (miRNAs) are small RNA molecules (~22 nt) that post-transcriptionally regulate the expression of protein-coding genes by mRNA degradation or translational repression. The functional miRNA form is derived from long primary transcripts via a series of maturation steps: miRNAs are transcribed as long primary transcripts (pri-miRNA) that undergo Drosha processing, releasing short hairpin precursor miRNAs (pre-miRNAs) which are subsequently cleaved by Dicer forming mature miRNAs that are loaded onto Argonaute. The mature miRNA guides Argonaute-containing miRNA induced silencing complexes (miRISC) to specific target sequences in protein-coding mRNAs via imperfect base-pairing interactions and this association results in translational repression and destabilization of the target mRNA.
Here we show that not only protein-coding mRNA transcripts, but also the primary transcript of
let-7 miRNA is bound and regulated by Argonaute-containing miRISC complexes. We produced a genome-wide map of interactions between Argonaute Like Gene 1 (ALG-1) and target transcripts in C. elegans and discovered an Argonaute-binding site downstream of the
let-7 precursor hairpin, towards the 3' end of the primary transcript. We validated these results with RNA immunoprecipitation assays, demonstrating that ALG-1 physically associates with
let-7 primary transcripts and that the ALG-1-binding site is essential for this interaction. Moreover, direct ALG-1 interaction with primary
let-7 leads to lower primary
let-7 levels, as indicated by the accumulation of
let-7 primary transcripts in animals lacking ALG-1 protein or its binding site. Interestingly, the mature
let-7 can pair to and regulate its own primary transcript: in worms harboring a point mutation in the mature
let-7 sequence, which disrupts pairing with the target site, the
let-7 primary transcripts no longer associate with ALG-1 and their expression levels are elevated. Furthermore, disruption of ALG-1 binding to
let-7 primary transcripts results in reduced mature
let-7 levels. Deletion of the ALG-1 binding site from primary
let-7 or a weakened pairing capacity of mature
let-7 with its own primary transcript is also associated with less pre-
let-7, suggesting a novel role for Argonaute in the maturation step from primary to precursor
let-7. Together, our results show that ALG-1 and
let-7 are involved in a positive feedback loop that promotes
let-7 biogenesis. This is the first report of Argonaute regulating a primary transcript guided by its own mature miRNA and reveals a new paradigm for the role of Argonaute in gene regulation, as well as a new model of direct miRNA auto-regulation.