D. melanogaster Pumilio-Nos-Hunchback RNA Complex

Alexandra Thoms and Erika Pontillo '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 (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


Importantly, upon binding to Nanos, the alpha helix of Pum unfolds to promote interactions of residues with the upstream RNA. Specifically, Thr 1415, Lys 1377, and Lys 1413 as they approach the phosphate groups O4 atoms on the backbone. This is what causes the decrease in sequence-specificity. Research shows that Nos can stabilize binding of Pum to RNAs containing a wider range of consensus or divergent sequences, as long as the sequence is not disrupted.



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.

It is important to the two different C-terminal regions of each protein. The C-terminal Pum region interacts with RNA, while the C-terminal Nos region interacts with Pum. The C-terminal of Pum undergoes notable changes. Loop residues between repeats R7 and R8 rearrange to promote Van der Waals the of Phe 1367 with the C-terminal alpha helix of Nos. With the deletion of its C-terminal end, Nos cannot bind to Pum nor the target mRNA. The mutant containing a Nos C-terminal deletion had a strong defect in abdominal segmentation in Drosophila, suggesting that this interaction is essential for in vivo activity of the proteins (Figure 4).

Recall from the previous section the C-terminal the of Pum. The Pum Glu 1363 side chain is in hydrogen bonding distance of backbone N and O atoms of the Ile 382. In addition, Pum Phe 1367 forms part of a that interacts with the Nos C-terminus Met 378.


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 (weidmann et al, 2016).

V. Nanos and RNA

Nanos binds to three nucleotides of the PRE. The sequence is less specific for Nos, as Nos binds based on Pumilio recognition; however, the hb protein Nos binding sequence is typically 5'-UAA-3'. The are essential for the Pum-Nos-RNA complex to form. They the RNA bases immediately upstream of the PRE and, together with the C-terminal region of Nos, embrace the RNA and Pum. The ternary complex has a higher affinity to the target mRNA than Pum alone (Figure 5) Both pieces of evidence suggests that Nanos acts as a molecular clamp in this complex.

The first base (-1U) is inserted into a formed by Phe 321, Thr 366, and Tyr 369. This brings the O4 atom of -1U into close to the main chain N-atom of Thr 366. Similar interactions happen with RNA bases -2A and -3A. A mutant of Lys 368 had decreased repression activity. This is because there is an important salt bridge between the amino acid and the -1U phosphate group.

Figure 5. A gel shift assay shows how Nos increases Pumilio's binding affinity to the hb RNA in D. melanogaster.The ternary complex formation (all three components) allows for Nanos to increase the binding affinity of the regulatory complex to RNA. (Weidmann et al, 2016).

VI. References

Barker, D. D., Wang, C., Moore, J., Dickinson, L. K., Lehmann, R. 1992. Pumilio is essential for function but not for distribution of the D. melanogaster abdominal determinant Nanos. Genes and Development 6:2312-2326.

Wang, C., Lechmann, R. 1991. Nanos is the localized posterior determinant in Drosophila. Cell 66:637-647.

Wang, X., McLachlan, J., Zamore, P. D., Hall, T. M. 2002. Molecular recognition of RNA by a human pumilio-homology domain. Cell 110:501-512.

Weidmann, C., Qiu, C., Arvola, R., Lou, T., Killingsworth, J., Campbell, Z., Hall, T., Goldstrohm, A. 2016. Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.Biophysics and Structural Biology | Developmental Biology and Stem Cells. DOI: 10.7554/eLife.17096.

Back to Top