Computational modeling of the Mei-P26 NHL interaction with its RNA substrate allowed us to predict and experimentally validate key residues critical for sequence-specific RNA binding. We confirm the high structural similarity between the NHL domains of Mei-P26 and Brat, but also detect subtle differences that influence their function and affect target specificity.
Finally, the NHL domains of both Brat and Mei-P26 interact with RNA in a sequence-specific manner ( 3, 6, 32).Ĭonsidering the functional importance of the Mei-P26 NHL domain, we conducted comprehensive structural and functional analyses. The Mei-P26 NHL domain is required for the interaction with at least some of these proteins, as mutations leading to substitutions in its sequence abolish the interaction with Ago1 and impair ovarian stem cell maintenance ( 21). Similarly, Mei-P26 function requires several additional proteins, among them, Sxl (Sex-lethal), Bam (Bag of marbles), Bgcn (Benign gonial cell neoplasm), Wuho, and Ago1 (Argonaute-1) ( 17, 21, 30, 31). The Brat NHL domain participates in multiple direct or RNA-mediated interactions involving factors such as Pum, eIF4EHP and Miranda as binding partners ( 23, 24, 25, 26, 27, 28, 29). The function of the closely related Brat protein critically depends on its NHL domain as its deletion or other sequence alterations result in strong phenotypes that can be partially rescued by expression of the NHL domain alone ( 22, 23). Moreover, ovarian cells lacking Mei-P26 activity grow abnormally large and exhibit increased nucleolar size ( 21). In the male germline, Mei-P26 limits mitotic divisions during the differentiation process of precursor germ cells preventing over-proliferation ( 16). It ensures maintenance of germline stem cells through control of BMP signaling ( 17), but also promotes differentiation of daughter cells upon exit from the stem cell niche ( 18). In the female germline, Mei-P26 supports both cellular programs. Germline homeostasis depends on the maintenance of germline stem cells in the stem cell niche and on the proper differentiation of their progeny into gametes ( 20). Mei-P26 was identified as a regulator of differentiation in the male and female germline and its loss results in over-proliferation of germline cells, tumor formation and sterility ( 15, 16, 17, 18, 19). The Drosophila melanogaster genome encodes several proteins with a TRIM-NHL-like architecture, among them Brain tumor (Brat) and Meiotic-P26 (Mei-P26). TRIM-NHL proteins play important roles in development where they control cell fate decisions to regulate differentiation and cell growth ( 11, 14). The NHL domain folds into a β-propeller that typically acts as a scaffold to mediate interactions with other biomolecules such as proteins, DNA or RNA ( 6, 12, 13). The TRIM-NHL protein family shares a common architecture that comprises an N-terminal tripartite motif (TRIM, consisting of a RING domain, one or two B-Box type zinc fingers and a coiled-coil domain) followed by a C-terminal NCL-1, HT2A, and LIN-41 (NHL) domain ( 7, 8, 9, 10, 11). Several members of the evolutionary conserved TRIM-NHL family use their NHL domains to interact with RNA ( 2, 3, 4, 5, 6). RBPs typically bind RNAs through dedicated RNA-binding domains (RBDs) ( 1). They comprise a large and functionally diverse group of proteins involved in all aspects of RNA biology from RNA synthesis to its degradation. RNA-binding proteins (RBPs) play key roles in the post-transcriptional regulation of gene expression.