Structure of 3'-end RNA processing complexes
Final Report Abstract
The scientific achievements of my lab made possible by the financial support from the DFG can be separated into three main aspects: Aspect 1: We, in collaboration with the Buratowski lab at Harvard Medical School, were the first who showed how temporal recruitment of non-coding RNA processing machines to RNA polymerase II is accomplished. We showed that recruitment requires a phosporylation pattern that is imprinted on the RNA polymerase II during early elongation. Furthermore, we have shown how Nrd1 and Nab3, the two main components of this machine, form a high affinity complex. We also elucidated the mechanisms for acquiring specificity to the nascent RNA and determined the structure of the RNA recognition motif of Nab3 in complex with RNA. We were further interested the structure/ function relationship of the third component of the non-coding RNA processing machinery, the helicase Sen1. Regrettably, we never got hands on suitable material for structural studies. However, we did successfully obtain material of DDX1 helicase, which has been proposed to be involved in mRNA and tRNA processing. We determined the crystal structure of its SPRY domain, which is a unique feature of this family of helicases. Additionally, we extensively characterized the ATP-hydrolysis kinetics and ATP / ADP and RNA binding properties by steady state and transient kinetics. In conclusion, our studies revealed that DDX1 must rely on auxiliary factors for functionality, since it predominantly exists in an “open”, non-processive ADP-bound form. Aspect 2: We are the first to provide a molecular movie of catalysis by the RNA-specific polynucleotide kinase Clp1, which is an important subunit of mRNA but also tRNA processing machines. We showed how specificity for RNA is achieved and provided a model, how auxiliary factors can regulate Clp1’s kinase activity. Furthermore, in bioinformatic studies, we could show that the mechanisms of acquiring specificity for RNA and enzyme catalysis are conserved among all known eukaryotic RNA specific polynucleotide kinases. Most importantly, we identified a hitherto unknown “gating” mechanism of the well known Walker A lysine residue of the ATP binding site that prevents any futile ATP hydrolysis. Moreover, we could show that “gated” Walker A lysines have been observed in previous structures, however, their function remained unidentified. Aspect 3: In a joint effort with our collaborators, we determined crystal structures of the representatives for all four different families of RNA polymerase II CTD interacting domains, some of them in complex with different phospho-variants of the CTD. These studies revealed how these different family members acquire specificity to the CTD and provided substantial insights how the “CTD-code” imprinted by the specific phosphorylation pattern is deciphered. Additionally, we could show that some of these members bind cooperatively to multiple repeats on the CTD and thereby augment affinity for RNA polymerase II. These studies allowed us to provide a comprehensive and detailed description of the structure/function relationship of the most abundant RNA polymerase II binding domain family.
Publications
- (2008) Snapshots of the RNA processing factor SCAF8 bound to different phosphorylated forms of the carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem. 283(33) 22659-22669
Becker, R., Loll, B., Meinhart, A.
- (2008) The Nrd1- Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain. Nat. Struct. Mol. Biol. 15(8) 795-804
Vasiljeva, L., Kim, M., Mutschler, H., Buratowski, S., Meinhart, A.
- (2010) Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat. Struct. Mol. Biol. 17(10) 1195-1201
Lunde, B.M., Reichow, S.L., Kim, M., Suh, H., Leeper, T.C., Yang, F., Mutschler, H., Buratowski, S., Meinhart, A., Varani, G.
- (2010) Coupled RNA polymerase II transcription and 3’ end formation with yeast whole cell extracts. RNA 16(11) 2205-2217
Mariconti, L., Loll, B., Schlinkmann, K., Wengi, A., Meinhart, A., Dichtl, B.
- (2011) Structural insights into cis element recognition of non-polyadenylated RNAs by the Nab3-RRM. Nucleic Acids Res. 39(1) 337-346
Lunde, B.M., Hörner, M., Meinhart, A.
- (2011) The P-loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in pre-mRNA 3’-end formation. PLoS One 6(12) e29139
Holbein, S., Scola, S., Loll, B., Dichtl, B.S., Hübner, W., Meinhart, A., Dichtl, B.
- (2014) RNA specificity and regulation of catalysis in the eukaryotic polynucleotide kinase Clp1. Molecular Cell Volume 54, Issue 6, 19 June 2014, Pages 975-986
Dikfidan, A., Loll, B., Zeymer, C., Magler, I., Clausen, T., Meinhart, A.
(See online at https://doi.org/10.1016/j.molcel.2014.04.005)
