Crystal structure of the HEAT domain from the Pre-mRNA processing factor Symplekin.

The majority of eukaryotic pre-mRNAs are processed by 3′-end cleavage and polyadenylation, although in metazoa the replication-dependant histone mRNAs are processed by 3′-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ~1,160-residue protein Symplekin. Secondary structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 A resolution using SAD phasing methods. The structure exhibits ​exhibits55 canonical HEAT repeats along with an extended 31 amino acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3′-end processing. Taken together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process. Figure 5 Symplekin structural alignment with the two most closely related structures. (a) Symplekin superimposed with H. sapiens Cand1 of the Cand1-Cul1-Roc1 complex (PDB 1u6g). Cand1 structure is in grey, Cul1 in white, and Roc1 is removed for figure clarity. ... Keywords: Crystal structure, molecular dynamics, protein scaffold, HEAT repeat, mRNA processing INTRODUCTION Maturation of most eukaryotic pre-mRNAs requires cleavage and polyadenylation of the 3′-ends of primary transcripts. The 3′-end polyA tail ensures proper translation by delivering ribosomes to the mRNA1; in amphibian oocytes, it was shown that translation was eliminated when the polyA tail addition was blocked by chemical modification2. The polyA tail is also essential for protecting the message from exonucleases and for transporting the message from the nucleus to the cytoplasm3. The length of the polyA tail affects the stability of the message, and compromised stability has been shown to lead to inflammation, cancer, early developmental maladies and coronary ailments4. Thus, proper polyA tail addition to messenger RNA is required for proper cellular function. For polyadenylation to occur, the cleavage stimulation factor (CstF) and the cleavage and polyadenylation specificity factor (CPSF) must work in concert to recognize and orient the cleavage site for the addition of the polyA tail5. The ~1,160 residue Symplekin protein is proposed to be the scaffolding factor on which this large protein complex is assembled3. Symplekin binds two members of the CstF macromolecular complex, CstF64 and CstF77, in a mutually exclusive manner6. Symplekin was identified as a stoichiometric component of the polyadenylation complex recently isolated from mammalian cells7. Symplekin, CPSF73, and CPSF100 are part of a stable complex in D. melanogaster as shown via co-immunoprecipitation and co-depletion studies8. Metazoan replication-dependent histone mRNAs are unique in that their 3′-ends are cleaved, but not polyadenylated. Interestingly, fractionation of HeLa cell nuclear extracts also identified Symplekin as a component of the histone pre-mRNA processing machinery9. Additionally, an extensive RNA interference (RNAi) screen found Symplekin to be necessary for histone pre-mRNA processing in D. melanogaster; when Symplekin was RNAi-depleted, a histone pre-mRNA reporter10 and endogenous histone mRNA8 was misprocessed. These data lead to the hypothesis that Symplekin is essential for proper 3′-end formation of canonical and histone mRNA by providing a scaffold on which protein-protein interactions can occur6,9. Symplekin may also serve as a bridging factor between the polyadenylation machinery and transcription regulators. Most recently, the N-terminal region of yeast Symplekin (Pta1) was found to interact with Ssu72, an RNA polymerase II C-terminal domain (CTD) serine 5-phosphatase11. The 124 N-terminal residues of mouse Symplekin interact with heat shock factor 1 (HSF1). HSF1, Symplekin and other polyadenylation factors coimmunoprecipitate with HSF1 after heat shock, leading to the suggestion that HSF1 stimulates both transcription and processing12. Over-expression of a non-DNA binding mutant of HSF1, which can sequester Symplekin, decreased Hsp70 mRNA polyadenylation in stressed cells12. Thus, the N-terminal region of Symplekin may be involved in protein-protein interactions that help couple transcription and processing. Utilizing in silico methods13–19, several potential HEAT repeats were identified in the N-terminus of D. melanogaster Symplekin. Protein domains formed by HEAT repeats are established protein-protein interaction scaffolds20–27. HEAT repeats are composed of 37–47 residues that fold into two anti-parallel helices connected by short (1–10 amino acids) linkers. Each set of helices can repeat 3 to 36 times, creating a HEAT domain16. To characterize the N-terminal region of the Symplekins, the three-dimensional structure of D. melanogaster Symplekin residues 19–271 was determined using SAD phasing and refined to 2.4 A resolution. Additionally, molecular dynamics simulations were employed to examine motion within this molecular scaffold. Taken together, these results provide the first detailed structural information on Symplekin, and indicate that the Symplekin HEAT domain may serve as a scaffold for protein-protein interactions essential to the mRNA maturation process.