%0 Generic
%A Falb, Melanie
%D 2009
%F heidok:10382
%K NMR , U4 snRNA , Kink-turn , lariat
%R 10.11588/heidok.00010382
%T NMR Structural Investigations of the U4 snRNA kink-turn and of a lariat-forming ribozyme
%U https://archiv.ub.uni-heidelberg.de/volltextserver/10382/
%X Three-dimensional fold of the U4 5’stem loop snRNA in its unbound form: The spliceosome, which catalyzes the splicing of eucaryotic pre-mRNAs, consists of five uridine-rich small nuclear ribonucleoprotein particles (U snRNPs) and numerous other factors. To activate the spliceosome and to enable the first step of splicing, the paired U4 and U6 snRNAs of the U4/U6 snRNP complex need to dissociate from each other. An initial step is the binding of the protein 15.5K to the 5’ stem loop of U4 snRNA (U4 5’ SL). Subsequently, further proteins are recruited, in particular protein hPrp31, which forms a ternary complex with the U4 5’ SL and 15.5K. Upon binding to the 15.5K protein, the U4 5’ SL folds into a characteristic structural motif, called the kink-turn (k-turn), in which the phosphodiester backbone presents a particular sharp turn. In this work the three-dimensional conformation of the free U4 5’ SL RNA in solution was investigated in order to elucidate whether the k-turn structural element is already present in the absence of protein binders.  By using NMR spectroscopy, the structure of the unbound U4 5’ SL was solved at a precision of 0.6 Å. In the structure, the canonical as well as the non-canonical stem of the U4 5’ SL present a well-defined helix fold. The U4 5’ SL lacks the characteristic sharp turn of the k-turn motif reported for the protein bound form. Instead, the free U4 5’ SL presents a more opened, extended conformation. The two non-canonical G-A base-pairs found in the k-turn structure are already formed in the unbound RNA, but the three unpaired residues of the internal loop (AAU) are stacked differently with respect to the k-turn motif.  In this work it was shown that the free U4 k-turn RNA is prevalently found in the extended conformation in solution. Thus, the consensus sequence of the k-turn does not per se code for the sharp bent in the RNA backbone. Instead, the structural k-turn element is highly disfavoured in solution and needs to be stabilized by protein binding, which favors the thesis of a protein-assisted mechanism for k-turn RNA folding.  Structural investigations of a spliceosomal related lariat-forming ribozyme: In eukaryotic cells, pre-mRNAs are processed by the spliceosome in a way that internal non-coding regions (introns) are excised and the remaining segments (exons) are joined together. The active spliceosomal core consists of paired U2 and U6 snRNAs, which juxtapose the splice site residues of the pre-mRNA substrate. While the RNA-RNA interactions during splicing have been well-characterized in the past, the description of tertiary structure of the spliceosomal catalytic center remains a challenge due to the large size and dynamics of the complex. In our laboratory a model ribozyme is investigated, which undergoes a transesterfication reaction with striking similarity to the first step of splicing by forming a 2’-5’ lariat. The lariat formation has the same sequence specificity, including the phylogenetically highly conserved ACAGAGA box that is essential for catalytic activity in the spliceosome of higher eukaryotes.  In this work, mutational studies were carried out to identify specific sites that can be either linked to folding of the ribozyme or to their function in the catalytic activity of the RNA molecule. With these studies it could be shown that the residues A29, G32, A33 and A35 are functionally the most important residues within the ACAGAGA-segment. Furthermore, a well defined helical moiety in the 5’ region of the ribozyme with an unusual high content of non-canonical base-pairs was revealed by combining information of the mutational studies with NMR derived distance constraints.  Another aim of this work was to gain first insights into the three-dimensional fold of the ribozyme prior to catalysis. NMR investigation and analysis were performed, with several differentially 13C, 15N-labeled NMR samples, in order to retrieve numerous complementary NMR spectra for the resonance assignment of the ribozyme. However, although various labeling schemes were applied, the assignment remained ambiguous for some nucleotides, due to both spectral overlap and a conformational exchange process that was detected for half of the resonances. To overcome the spectral complexity, eight RNA mutants were constructed, which enabled a complete, unambiguous resonance assignment of all nucleotides of the lariat-forming ribozyme. This resonance assignment is a perquisite for the collection of structural restraints, mainly of NOE peak intensities. NOE signals reflect direct distances of neighboring atoms in a molecule and suggest a compact fold, presumably containing both a ribose zipper motif and a pseudoknot motif, for the lariat-forming ribozyme. All these data represent an excellent starting point to explore the complete three-dimensional structure of the lariat-forming ribozyme and will facilitate its understanding in terms of functionality.