Biochemical and Single Molecule Studies of Backbone Branched RNAs and Lariat Debranching Enzyme

The intronic lariat RNA generated during pre-mRNA splicing includes a branch-point adenosine residue that is linked through the 2'-O position to the 5'-end of the RNA sequence. This 2'-5'-phosphodiester linkage in the lariat RNA backbone is cleaved (debranched) by the lariat debranching enzyme (Dbr1p). Following this debranching reaction, some introns can participate in highly important biological processes like snoRNA biogenesis, microRNA pathways etc. Although Dbr1p has structural resemblance to some non-specific nucleases, it still remains elusive how Dbr1p can selectively cleave the 2'-5' phosphodiester bond in lariat RNAs without affecting the 3'-5' phosphodiester bonds. The major roadblock towards understanding Dbr1p mechanism has been easy access to its substrate. We developed a branching phosphoramidite with 3'-photoprotecing group which gives us facile access to 2'-5' phosphodiester linked backbone branched RNAs (bbRNAs) which are mimics of the lariat RNAs. Our method of bbRNA synthesis is versatile and allows incorporation of both internal and terminal modifications. To demonstrate this, several bbRNAs with modified 2'-branch were synthesized using this method which showed different debranching activity. To examine the kinetics of the debranching reaction, we synthesized a dual-fluorescently-labeled bbRNA with Cy3 and Cy5 dyes that acts as donor and acceptor in a FRET based assay. By following the increase in the donor emission with time for this dual-labeled substrate, the debranching reaction can be followed in real time. Using this assay we have been able to find the kinetics parameters of the Dbr1p enzyme for the first time. We have also synthesized a non-cleavable analogue of the native bbRNA substrate where the 2'-5' phosphodiester bond is replaced by a triazole (click) linkage. Using the FRET based kinetics we show that this ‘click’ branched RNA (cbRNA) is a competitive inhibitor of Dbr1p. This suggests that the cbRNA binds the Dbr1p enzyme in a similar fashion as the native substrate. The unique structure of 2'-5' phosphodiester linked bbRNAs could play an important role in the selective cleavage of bbRNA substrates by Dbr1p. While synthetic access to bbRNAs has enabled biochemical and kinetics studies of the debranching reaction, these studies do not inform regarding the structure and dynamics of the bbRNAs. Towards this goal, we performed single molecule FRET (smFRET) experiments using dual-labeled (Alexa488 Alexa594) bbRNA and its non-cleavable analogue, cbRNA. SmFRET experiments using confocal microscopy revealed a single broad distribution of cbRNA conformations, whereas the native bbRNAs showed two distinct populations. For both species, addition of divalent metal ions (Mn2+ and Mg2+) induces a high FRET population indicating that the stem and the branch strands of the bbRNA are in close proximity. Additionally, conversion of the stem to a duplex gives rise to a single narrow FRET distribution meaning that both the native bbRNA and cbRNA lose conformational flexibility. Finally, using TIRF microscopy, we showed that cbRNAs undergo a conformational switch between a high and a medium FRET state. However the importance of these conformations and their fluctuations, for the debranching reaction remains to be investigated. Together this two-pronged approach of biochemical and single molecule studies will help the reaction mechanism of the elusive lariat debranching enzyme to be elucidated in the future.