The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.
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 β 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.
Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Inga
I
Loedige
Mathias
M
Stotz
Saadia
S
Qamar
Katharina
K
Kramer
Janosch
J
Hennig
Thomas
T
Schubert
Patrick
P
Löffler
Gernot
G
Längst
Rainer
R
Merkl
Henning
H
Urlaub
Gunter
G
Meister
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C412783:brat protein, Drosophila; C061245:hb protein, Drosophila; C074333:pum protein, Drosophila
BRAT; RNA binding; TRIM-NHL; gene regulation; hunchback; translational repression