D. melanogaster Pumilio-Nos-hunchback RNA complex

Erika Pontillo & Alex Thoms '23

Contents:

I. Introduction

Pumilio (Pum) and Nanos (Nos) are RNA-binding proteins that work together as a combinatorial translational regulatory complex. These proteins influence development, the nervous system, and the behavior of stem cells.

In Drosophila, they are responsible for the repression of maternal hunchback (hb) RNA. The hb gene has multiple functions early in development, however, its function must be repressed for some body structures to form, such as abdominal segments. The activity of the Pum-Nos repressing complex is sequence-specific, they recognize a particular domain that is conserved in Pum regulated genes. Genome-wide analyses have identified hundreds of Pu associated mRNAs, suggesting that Pum may regulate much more than a few validated genes. The initial recognition of RNA and binding of the complex is done by Pum, Nanos acts as a molecular clamp that regulates and modulates the RNA-binding and repression activities of Pumilio.

In the absence of Pum and Nos expression, the hb protein in Drosophila is expressed throughout the embryo and results in no abdominal segment formation. This molecular model demonstrates how cooperative RNA binding proteins regulate gene expression.

A of the Pum-Nos-RNA complex demonstrates some interesting interactions between the three components. Nos interacts with Pum and RNA, adds to the sequence-specific contacts, and increases the binding affinity of Pum. Nos also shifts the recognition sequence and promotes a repression complex formation on the mRNA (Figure 1). The Pum-mRNA complex is not stably bound alone.


Figure 1. The Pum and Nos RNA-binding proteins have some sequence specificity that is adjusted when interactions between the proteins occur. (Weidmann et al, 2016)

II. General Structure

It is important to understand how the different structures work together on this molecular model. The amino acids side chains on the proteins and bind to bases in the target .

Pumilio interacts with the RNA in a manner. Both Nanos and Pumilio also have interactions once attached to the RNA chain. It is the of the Nanos proteins that interact with Pumilio to repress target gene expression.

In our example, the hunchback RNA has the Pum-binding sequence, the binding between these two components has a code (Section III) that can be engineered to bind any 8 nucleotide sequence. Nanos then joins the complex. This smaller protein increases the binding affinity of Pum by decreasing its sequence specificity. Effector molecules are then recruited to the complex that remove the poly-A tail and repress translation of huncback mRNA in the posterior abdomen of Drosophila.


Figure 2. The Nos protein binding to Pumilio alters the sequence specificity of the complex while also increasing the proteins affinity. (Hall Presentation at Kenyon College, 2020)

III. Pumilio and RNA

Pumilio form a curved alpha-helical domain. It can be appreciated that the mRNA binds to the concave portion of the protein, creating a domed binding domain on the mRNA strand. These "PUF" protein amino acid side chain repeats interact with the bases of the mRNA in a following a code (Figure 3). Certain 3-amino acid sequences in the loops connecting the alpha helices repeats bind to one of the four bases (Table 1). The consensus Pum-binding RNA sequence is 5'-UGUAHAUA-3' (where H can be A,U, or C). This sequence is referred to as the PRE (Pumilio response element). A Pum-regulated mRNA may have multiple PREs. Each repeat interacts with a single base on the mRNA strand. One side chain from the repeat interacts with one base in a hydrogen-bonding interaction. Often another side chain from a different Pum repeat is also involved in a stacked interaction.

Table 1. The amino acid and base binding involved in the Pumilio-RNA complex. The side-chain bonding interactions are either Hydrogen bonds or stacking. The combination of the two result in high binding energy.

RNA Base Amino Acids Involved                  
Interactions 
View 
Uracil
(U)		 Gln, Asn, Tyr, Asn
 Hydrogen Bonding, Stacking, Van Der Walls
Adenine
(A)		 Gln, Cys, Arg, Tyr

 Hydrogen Bonding, Stacking, Van Der Walls
Guanine
(G)		 Glu, Ser, Asn, Tyr
 Hydrogen Bonding, Stacking, Van Der Walls
Cytosine
(C)		 Arg, His
 Hydrogen Bonding, Van Der Walls




Figure 3. The Pumillio protein of D. melanogaster and FBF protein of C.elegans (PUF proteins) amino acid side chains bind to the mRNA bases the same way with each base. Van Der Walls interactions are subjected to change based on the sequence of the mRNA. (Wang et al, 2002)

IV. Pumilio and Nanos

Nanos and Pumilio interactions are what drive effective translational repression. Nos binding to hb requires Pum-RNA recognition. The addition of Nos at the upstream nucleotides induces localized conformational changes in Pum that promote Pum-Nos interactions and binding of Pum to the RNA upstream to the core PRE. The C-terminal of Pum undergoes notable changes. Loop residues between repeats R7 and R8 rearrange to promote of Phe 1367 with the C-terminal alpha helix of Nos. Phe 1367 and the C-terminal end of Nos have Van Der Waals .With the deletion of the C-terminal end of Nos, the remaining molecules of the protein cannot bind to Pumnor the mRNA making. The mutant containing a Nos C-terminal deletion had a strong defect in abdominal segmentation of Drosophila, suggesting that this interaction is essential for in vivo activity of the proteins (Figure 4).

Glu 1363 and Phe 1367 abrogates ternary complex formation, aligning the protein for proper binding with Pum. Ile 382 and Met 378 of the Nos protein interact with Pumilio alpha-helices through covalent bonding.


Figure 4. The hb (hunchback) protein is constitutively expressed in D.melanogaster when the C-terminal end of Nos is deleted in vivo via mutation. No abdominal segments are present in the embryo as the Nos-Pum complex is responsible for the gene repression in the posterior end of the organism (Tautz et al, 1988).

V. Nanos and RNA

Transcription activation by CAP requires more than merely the binding of cAMP and binding and bending of DNA. CAP contains an "activating region" that has been proposed to participate in direct protein-protein interactions with RNA polymerase and/or other basal transcription factors. Specifically, amino acids 156, 158, 159, and 162 have been proposed to be critical for transcription activation by CAP. These amino acids are part of a surface loop composed of residues 152-166 Researchers have concluded that the third and final step in transcription activation is this direct protein-protein contact between amino acids 156-162 of CAP, and RNA polymerase.


VI. References

Gunasekera, Angelo, Yon W. Ebright, and Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of the Escherichia coli Catabolite Gene Activator Protein. The Journal of Biological Chemistry 267:14713-14720.

Schultz, Steve C., George C. Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex: The DNA Is Bent by 90 degrees Science 253: 1001-1007.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

Back to Top