Table 1 Similarities and differences between the targeting rules for miRNA-guided fly AGO1 and mammal AGO2

Seed pairing

Seed complementarity dominates DmAgo1 target binding

Canonical seed pairing is the most efficient way to reach high-affinity binding

This study^23,24,25,53

G:U pairs in the seed

DmAgo1 does not tolerate G:U wobble pairs

AGO2 can display moderate affinity

This study^23,24,52

1-nt mismatches in the seed

Limited tolerance for mismatches, but some miRNAs may bind sites containing one single mismatch

Broader, the imperfections tending to occur at different positions, with affinities similar to those of the canonical sites

This study^23,24,52

1-nt bulges in the seed

Some nucleation-bulged sites are allowed

More diversity in bulged sites

This study^23,24,55

3′ pairing

Required for stable interaction with mismatched seeds

Stabilizes pairing with weak or imperfect seeds

This study^23,35

3′-only sites

Some 3’-only sites bind with comparable or higher affinity than the canonical 6mer site

Some 3’-only sites bind with comparable or higher affinity than the canonical 6mer site

This study^24,25,74

Centered sites

Can be cleaved by Ago1

Can be cleaved by AGO2

This study²⁶

Identity of nucleotides flanking binding sites

Impacts binding affinity, likely by influencing site accessibility

Impacts k_on and binding affinity, likely by influencing site accessibility

This study^23,24,25,62