The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

Abstract

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed b propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

The TRIM-NHL Protein Brat Promotes Axon Maintenance by Repressingsrc64BExpression

“…Although src64B mRNA contains a Pumbinding motif, its regulation by Brat is thus likely independent of Pum activity. This is consistent with recent work indicating that translation repression by Brat can occur independently of Pum in Drosophila S2 cells, and that Brat binds RNA independently of Pum, via its NHL domain (Loedige et al, 2014). Notably, similar conclusions were drawn with Brat mammalian ortholog TRIM71 (Loedige et al, 2013).…”

“…Second, we tried to rescue brat phenotypes by expressing a form of Brat that cannot associate with the Pum/Nos/hunchback NRE complex (Brat G774D ; Sonoda and Wharton, 2001; Harris et al, 2011). Although the G774D mutation was initially thought to disrupt a putative Pum-Brat interaction (Sonoda and Wharton, 2001), it was recently proposed to rather prevent the Pum-enhanced association of Brat to RNA (Loedige et al, 2014). Strikingly, expression of Brat G774D was able to rescue brat 192 Nb clone phenotype (Fig.…”

“…Although it had initially been proposed that the RNA binding protein Pum serves as a platform to recruit Brat and Nos to target RNAs (Sonoda and Wharton, 2001), it was recently demonstrated that Brat directly binds to the Nanos Response element (NRE) of hunchback 3ЈUTR (Loedige et al, 2014). Notably, Brat recognizes a sequence (BoxA motif) that flanks the Pum binding site (Fig.…”

“…6A). Furthermore, the recruitment of Brat is facilitated by Pum binding within the hunchback NRE (Loedige et al, 2014). To identify potential Brat targets, we analyzed the sequence of mRNAs encoding positive regulators of the previously described Rhodependent pathway.…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…Mutation of a specific cross-linking site might not completely abolish RNA-binding, as the RNA-binding region is larger than a single amino-acid residue. Such investigations had been performed on T. pendens protein Cas7 [24] or on the NHL domain (WD40 domain) of BRAT bound to RNA [55]. The protein-RNA cross-linking approach described here and in related studies [25] also addresses changes in binding of RNA to proteins in dependence upon different cellular environments and identifies transient interactions of the RNA with the proteins.…”