MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by repressing translation of target mRNAs. A study by Doench and Sharp reveals that the ability of an miRNA to repress translation depends primarily on the free energy of binding of the first eight nucleotides in the 5' region of the miRNA. However, G:U wobble base-pairing in this region can interfere with activity beyond what is predicted by thermodynamic stability. The study also shows that an mRNA can be repressed by multiple miRNAs, and the level of repression depends on the amount of mRNA and available miRNA complexes. The research highlights the importance of the 5' region of the miRNA in translational repression, with the 3' region playing a less critical role. However, the 3' region can modulate the effect of the 5' region. G:U wobble base pairs, which are thermodynamically favorable, are detrimental to miRNA function despite their favorable contribution to RNA:RNA duplexes. The study also demonstrates that miRNAs can simultaneously repress a single mRNA, indicating combinatorial regulation is a common feature of eukaryotic cells. The study used experimental approaches to investigate miRNA:mRNA interactions, including luciferase assays and ribonuclease protection assays. The results show that the free energy of the first 8 nt of the miRNA binding to the mRNA is a key determinant of repression. The study also confirms that the 5' region of the miRNA is more important for specificity and activity than the 3' region. The findings suggest that computational methods for predicting miRNA targets should consider the thermodynamic stability of the miRNA:mRNA interaction, as well as the potential for multiple miRNAs to target the same mRNA. The study also shows that the level of miRNA expression and the presence of competing binding sites on other mRNAs can influence whether an mRNA is endogenously regulated by a miRNA. The results emphasize the importance of considering the cellular context when evaluating miRNA target selection. The study provides insights into the specificity of miRNA target selection in translational repression, highlighting the role of the 5' region of the miRNA and the impact of G:U wobble base pairs on miRNA function.MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by repressing translation of target mRNAs. A study by Doench and Sharp reveals that the ability of an miRNA to repress translation depends primarily on the free energy of binding of the first eight nucleotides in the 5' region of the miRNA. However, G:U wobble base-pairing in this region can interfere with activity beyond what is predicted by thermodynamic stability. The study also shows that an mRNA can be repressed by multiple miRNAs, and the level of repression depends on the amount of mRNA and available miRNA complexes. The research highlights the importance of the 5' region of the miRNA in translational repression, with the 3' region playing a less critical role. However, the 3' region can modulate the effect of the 5' region. G:U wobble base pairs, which are thermodynamically favorable, are detrimental to miRNA function despite their favorable contribution to RNA:RNA duplexes. The study also demonstrates that miRNAs can simultaneously repress a single mRNA, indicating combinatorial regulation is a common feature of eukaryotic cells. The study used experimental approaches to investigate miRNA:mRNA interactions, including luciferase assays and ribonuclease protection assays. The results show that the free energy of the first 8 nt of the miRNA binding to the mRNA is a key determinant of repression. The study also confirms that the 5' region of the miRNA is more important for specificity and activity than the 3' region. The findings suggest that computational methods for predicting miRNA targets should consider the thermodynamic stability of the miRNA:mRNA interaction, as well as the potential for multiple miRNAs to target the same mRNA. The study also shows that the level of miRNA expression and the presence of competing binding sites on other mRNAs can influence whether an mRNA is endogenously regulated by a miRNA. The results emphasize the importance of considering the cellular context when evaluating miRNA target selection. The study provides insights into the specificity of miRNA target selection in translational repression, highlighting the role of the 5' region of the miRNA and the impact of G:U wobble base pairs on miRNA function.