From Proteopedia

Introduction

Hrp1 is a polyadenylation factor found in Saccharomyces cervisiae (yeast) ^[1]. This protein recognizes and binds to an RNA sequence in the 3'UTR of the messenger RNA (mRNA) upstream from the cleavage site called the polyadenylation enhancement element (PEE) ^[1]. Upon binding to the RNA, Hrp1 helps recruit additional proteins necessary for the cleavage and polyadenylation of the RNA molecule ^[1]. The unique structural features of the Hrp1-PEE complex reveals the mechanism by which Hrp1 is able to recognize and bind to its specific RNA sequence at the atomic level ^[1].

Structure

Hrp1 is a single strand RNA-binding protein composed of two RNP-type RNA-binding domains (RBDs) arranged in tandem with a typical ßαßßαß architecture ^[1]. The two RBDs have similar topolgies, both containing a central antiparallel four-stranded with two α-helices running across one face ^[1]. The two RBDs associate to form a deep and positively charged , which constitutes the binding site for the RNA molecule ^[1].

Figure 1: Cartoon representation of the Hrp1-PEE complex. The RNA is shown as a stick model and is colored by element. Notice the interface between the ß-sheets of Hrp1 and the RNA.

Hrp1-RNA Interactions

The interface between Hrp1 and its target RNA sequence is dominated by interactions between key aromatic residues and RNA nucleobases ^[1]. Only six RNA bases, an repeat, act as the PEE and form specific contacts with Hrp1 ^[1]. Hydrophilic residues of Hrp1 provide base specificity through hydrogen bonding ^[1]. Most of the key residues that interact with the RNA can be found in the ß-sheet region of Hrp1; however, loops and the interdomain linker are also essential for Hrp1-RNA recognition ^[1]. Perhaps the most important Hrp1-RNA interaction is the (a conserved residue) ^[1]. In this case, Trp168 stacks on Ade4 and forms crucial base-specific hydrogen bonds ^[1]. It is also worth noting that a second Hrp1 residue is critical to holding Ade4 in place, , which interacts via hydrogen bond with the N1 of Ade4 ^[1]. A third contributor, , also stacks with Ura7 to aid in RNA recognition and binding ^[1].

RBD-RBD Interactions and the Linker Region

As mentioned above, Hrp1 is composed of two RBDs. The RBDs are connected by a (a short two-turn α-helix) which also contains an crucial residue for RNA binding. Ile234 holds Ade6 stacked in place with Phe162 . Experimental evidence from the NMR data ^[1] suggests that the two RBDs at independently until binding the PEE. Binding the PEE causes the linker region to adopt a short helical structure to rigidly hold the . Aside from the linker helix, the only interaction between the RBDs is due to between Lys231 and Asp271 ^[1]. </StructureSection>

Figure 3: Interaction between Hrp1 (red), RNA15 (green) and RNA (blue).

Interaction with RNA15

RNA15 is another RNA-binding protein with a single N-terminal RNA recognition motif (RRM) ^[2]. RNA15 recognizes an A-rich positioning element (PE) downstream from the PEE but upstream from the 3' cleavage site ^[2]. The recognition of the PE by RNA15 is crucial for precise cleavage of the RNA molecule. Hrp1 and RNA15 are held together by a separate protein, RNA14 ^[2]. These proteins act together to anchor the polyadenylation and cleavage protein machinery relative to the cleavage site for precise 3'-end processing ^[2].

Figure 2: Sequence logo for residues 167-169 of Hrp1. The logo displays the frequency of residues occuring at specific positions within Hrp1. W168 is always conserved in Hrp1 and RRMs of similar proteins.

Relationship to other proteins

The RNP-type RBD is found in many proteins involved in post-transcriptional pre-mRNA processing (5'-end capping, splicing, 3'-end cleavage and polyadenylation, and transport from the nucleus)^[3]. The unique RBD of Hrp1 enables the protein to bind an RNA sequence that differs in both length and content from the RNA sequences of other RNA-binding and mRNA processing proteins such as sex lethal, Poly (A)-binding protein (PABP), and HuD ^[1]. Like Hrp1, each of these proteins belong to the class of single strand proteins composed of two canonical RBDs; however, these proteins are differentiated by their target RNA sequence, their interactions with RNA at the atomic level, and their interdomain contacts ^[1]. One way in which Hrp1 differentiates itself from these other proteins is by the fact that Hud, sex lethal, and PABP all contain at least one intra-RNA base-base stacking interaction, a feature that is not found in the Hrp1-PEE complex ^[1]. It is possible that the intra-RNA interactions found in these other proteins is replaced by the crucial Trp168-Ade4 stacking interaction found in the Hrp1 complex ^[1]. The fact that the intra-RNA base-base stacking interactions are replaced by the Trp168-Ade4 in the Hrp1-PEE complex might also explain why the Hrp1-RNA interface involves only 6 nucleotides whereas PABP, sex lethal, and HuD require a longer 8-10 nucleotide sequence in the RNA binding pocket ^[1].

References

1. ↑ ^{1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 1.21} Perez-Canadillas JM. Grabbing the message: structural basis of mRNA 3'UTR recognition by Hrp1. EMBO J. 2006 Jul 12;25(13):3167-78. Epub 2006 Jun 22. PMID:16794580
2. ↑ ^{2.0 2.1 2.2 2.3} Leeper TC, Qu X, Lu C, Moore C, Varani G. Novel protein-protein contacts facilitate mRNA 3'-processing signal recognition by Rna15 and Hrp1. J Mol Biol. 2010 Aug 20;401(3):334-49. Epub 2010 Jun 19. PMID:20600122 doi:10.1016/j.jmb.2010.06.032
3. ↑ Clery A, Blatter M, Allain FH. RNA recognition motifs: boring? Not quite. Curr Opin Struct Biol. 2008 Jun;18(3):290-8. doi: 10.1016/j.sbi.2008.04.002. PMID:18515081 doi:http://dx.doi.org/10.1016/j.sbi.2008.04.002