The role of pairing beyond the seed in microRNA targeting

MicroRNAs (miRNAs) are short non-coding RNAs which act as post-transcriptional regulators of gene expression. MiRNAs complex with Argonaute proteins to form an RNA-guided silencing complex (RISC). The guide RNA recognizes specific mRNAs via base-pairing to complementary target sites and typically induces repression of the gene product. In animals, target recognition is primarily mediated by the seed region, comprising the first eight nucleotides of the miRNA, but pairing outside the seed (3'-pairing) is sometimes required to achieve substantial repression. The determinants for effective 3'-pairing are not fully understood, limiting the accuracy of computational target site predictions.

To determine the precise base-pairing interactions in miRNA-target complexes, we developed RNA-RNA binding by SHAPE (RABS) (Paper I). We used this technique together with affinity measurements and reporter assays to obtain detailed information about the biochemistry of miRNA-target complexes with a variety of secondary structures, using the conserved miR-34a as a model (Paper II). Our results suggest that Argonaure modulates the affinity of the miRNA for its target sites in two directions, strengthening weak RNA:RNA binders and weakening strong ones, but the affinity is only weakly correlated with the amount of repression in cells.

We further explored the combined impact on site efficiency of 3'-pairing and secondary structures in the mRNA, present prior to miRNA binding or formed within the miRNA-target complex (Paper III). Using structural probing of miRNAtarget interactions by RABS and reporter assays to measure target repression in cells, we showed that 3'-pairing can compensate for decreased seed binding due to self-pairing in the mRNA. This enables downregulation of sites which would be non-functional if only seed pairing was available.

Finally, we modified nucleotides in miR-34a to disrupt pairing beyond the seed, enabling high-throughput screening of target sites for effective 3'-pairing in cells and subsequent identification of favourable and unfavourable structural features (Paper IV). We found that miR-34a is differentially sensitive to GU wobble pairs depending on their position in the 3'-pairing helix. It also prefers unpaired nucleotides on the miRNA side over the target site, in contrast with what has previously been observed for other miRNAs. This adds to a growing body of evidence that 3'-pairing preferences vary between different miRNAs.

List of scientific papers

I. Banijamali E, Baronti L, Becker W, Sajkowska-Kozielewicz JJ, Huang T, Palka C, Kosek D, Sweetapple L, Müller J, Stone MD, Andersson ER, Petzold K. (2023). RNA:RNA interaction in ternary complexes resolved by chemical probing. RNA. 29: 317-329.
https://doi.org/10.1261/rna.079190.122

II. Sweetapple L, Kosek DM, Banijamali E, Becker W, Müller J, Karadiakos C, Baronti L, Guzzetti I, Schritt D, Chen A, Andersson ER*, Petzold K*. (2024). Biophysics of microRNA-34a targeting and its influence on down-regulation. bioRxiv. *Co-corresponding authors. [Manuscript]
https://doi.org/10.1101/2024.02.14.580117

III. Kosek DM, Banijamali E, Becker W, Petzold K*, Andersson ER*. (2023). Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility. Nucleic Acids Research. 51: 11162–11177. *Co-corresponding authors.
https://doi.org/10.1093/nar/gkad795

IV. Kosek DM, Petzold K, Andersson ER. Mapping effective microRNA pairing beyond the seed using abasic mutations. [Manuscript]