Extending the L1 region in canonical double-stranded RNA-binding domains impairs their functions

A large number of proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), whose sequence variations and functional versatilities are still being recognized. All dsRBDs have a similar structural fold: alpha 1-L1-beta 1-L2-beta 2-L3-beta 3-L4-alpha 2 (alpha represents an alpha-helix, beta a beta-sheet, and L a loop conformation between the well-defined secondary structures). Our recent work revealed that the dsRBD in Drosha, which is involved in animal microRNA (miRNA) biogenesis, differs from other dsRBDs by containing a short insertion in its L1 region and that this insertion is important for Drosha function. We asked why the same insertion is excluded in all other dsRBDs and proposed that a longer L1 may be detrimental to their functions. In this study, to test this hypothesis, we inserted the Drosha sequence into several well-known dsRBDs from various organisms. Gel mobility shift assay demonstrated that L1 extension invariably reduced RNA binding by these dsRBDs. In addition, such a mutation in Dicer, another protein involved in miRNA biogenesis, impaired Dicer's ability to process miRNAs, which led to de-repression of reporter expression, in human cells. Taken together, our results add to the growing appreciation of the diversity in dsRBDs and suggest that dsRBDs have intricate structures and functions that are sensitive to perturbations in the L1 region.

Automated summary

Proteins involved in RNA metabolism possess a double-stranded RNA-binding domain (dsRBD), with variations in sequences and functions. A study showed that a short insertion in the dsRBD of Drosha is important for its function, and extending the L1 region in other dsRBDs reduces RNA binding. This highlights the sensitivity of dsRBDs to perturbations in the L1 region and adds to the understanding of their structural and functional diversity.