Determination of the structure of the RNA complex of a double-stranded RNA-binding domain fromDrosophila Staufen protein

Abstract

We have determined using NMR the structure of the complex between the third double‐stranded RNA‐binding domain (dsRBD3) of Drosophila Staufen protein and a RNA stem‐loop with optimal binding properties in vitro. This work was designed to understand how dsRBD proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA–protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD–RNA binding and to the physiological role of Staufen protein.©2001 John Wiley & Sons, Inc. Biopoly( Nucleic Acid Sci) 52: 181–196, 1999/2000

Solution structure of the Drosha double-stranded RNA-binding domain

“…Typical RNA-interacting regions are indicated with brackets, and RNA-interacting residues are in bold. Sequences of Aquifex aeolicus RNase III (AaRnIII) [24], Saccharomyces cerevisiae Rnt1p [23,35], Xenopus laevis Xlrbpa-2 [26], Drosophila melanogaster Staufen dsRBD-3 [25], and DiGeorge syndrome critical region 8 (DGCR8) protein dsRBD 1 (DGCR8-1) and 2 (DGCR8-2)[19] are shown. (d, e) Electrostatic surface representation calculated using the APBS package [36] of the lowest energy-minimized structure of Drosha-dsRBD.…”

A multipronged approach to understanding the form and function of hStaufen protein

“…dimensional models of truncation mutants, in isolation or in complex with short RNA sequences or truncated protein interactors (Ramos et al 1999(Ramos et al , 2000Gleghorn et al 2013;Gleghorn and Maquat 2014;Jia et al 2015;Lazzaretti et al 2018) made its functional understanding particularly challenging. Here, we provided for the first time structural information on the full-length hStau1 protein, using an integrated structural biology approach.…”

“…The solution structure of Drosophila melanogaster Stau RBD3 bound to a 12-bp stem-loop RNA, determined by NMR spectroscopy, revealed the interaction of the canonical α-β-β-β-α RBD fold with dsRNA (PDB ID: 1EKZ) (Ramos et al 1999(Ramos et al , 2000. The crystallographic structure of the RBD3-RBD4 construct, bound to dsRNA as a dimer (monomers A and B), shows that the interaction surface with the RNA spans the major groove and the two adjacent minor groove surfaces.…”

Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein

“…By contrast, paramagnetic applications to RNA suffer from issues associated with this direct chemical tagging, including sample instability and low tagging efficiency. Although techniques are available to incorporate modified bases in a site-specific manner, − these often involve relatively low-yielding multistep enzymatic or synthetic protocols. Perhaps the biggest challenge is the inability to tag specific sites in large RNAs prepared by in vitro transcription.…”