Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins

Abstract

Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control.

Keywords: Brain Tumor (or Brat); Nanos; Pumilio; combinatorial mRNA regulation; cooperative RNA binding.

Figure 1.

Pum, Nos, and Brat are RNA-binding proteins that bind the hunchback mRNA. (A) Schematic diagrams of Pum, Nos, and Brat proteins with relevant domains labeled: Pum N-terminal Repression Domains (RD1, RD2, and RD3), and Pum Homology Domain (Pum-HD); Nos Effector Domain (NED), Zinc Fingers (Z), and C-terminal extension (C); Brat B-box Zinc Fingers 1 and 2 (B1 and B2), coiled coil (CC), and NCL^-1, HT2A, and Lin-41 (NHL) domain. (B) Pum, Nos and Brat bind to the Nanos Response Element 2 (NRE2) RNA from the hunchback 3´UTR with color-coded binding sites for Brat, Nos, and Pum. Box A and B elements of the NRE are outlined by a black box. Direct interactions are indicated by solid lines whereas dashed lines indicate putative interactions. The loop between repeats 7 and 8 of Pum, which mediates protein-protein interaction with Nos, is shown in black. (C) Structural model of Brat (NHL domain), Nos (ZC regions), and Pum (Pum-HD) proteins with NRE RNA. The crystal structures of Brat in complex with a BBS (red, PDB ID 4ZLR) and Nos (blue)/Pum (yellow) in complex with NBS-PRE RNA (PDB ID 5KL1) are shown with the 4 nucleotide spacer RNA (gray) present in the native hunchback NRE2 RNA. Brat and Pum proteins are shown as ribbon diagrams. Nos is shown with a molecular surface superimposed. Residue G1330 of Pum is highlighted by a yellow sphere.

Figure 2.

Multiple mechanisms of repression by Pum, Nos, and Brat. (A) Translational repression of target mRNAs can be mediated through recruitment of alternative cap-binding protein 4EHP by Brat, which is proposed to prevent binding of eIF4F translation initiation complex to the 5´ cap structure (7-methyl guanosine 5´-5´ triphosphate, m7Gppp). In addition, Pum antagonizes the translation activity of Poly(A) binding protein (PABP). (B) mRNA decay can be initiated through recruitment of the Ccr4-NOT (CNOT) complex, which catalyzes deadenylation and promotes decapping of the target mRNA. Pum recruits the Pop2 deadenylase to stimulate deadenylation, and Nos directly recruits Not1 and Not3 of the CNOT complex to stimulate deadenylation and decapping. Solid lines indicate documented interactions whereas dashed lines indicate putative interactions.

Figure 3.

Classification of Pum, Nos, and Brat target mRNAs. Classification of target mRNAs regulated by Pum, Nos, and/or Brat, based on experimental evidence and bioinformatics analysis of Drosophila 3´UTRs using RNA-binding affinity, specificity, and cooperativity. Brat Binding Site (BBS), Pum Response Element (PRE), relaxed PRE (rPRE), and Nos Binding Site (NBS), described in the text, are indicated for each of 6 categories.