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Department of Bioorganic Chemistry


Studies on the synthesis and structure of lariat-RNAs modelling the processing of pre-mRNA (Splicing)



Introduction

Why is Lariat-RNA formed in the RNA splicing reaction?

Both in the nuclear pre-mRNA- and in the Group II self-splicing reaction, introns splice out by way of two successive transesterification reactions (Fig. 1). The cleavage at the 5'-junction in splicing is due to an intramolecular nucleophilic attack by a 2'-OH from within the intron. This creates a lariat-RNA intermediate (Fig. 1) with the 5'-end of the intron attached through a 2'→5'-phosphodiester bond to a residue near the 3'-end of the intron in the penultimate step of the splicing reaction (Fig. 1). Subsequent cleavage at the 3'-junction results in the ligation of the exons and liberates the "free" intron in the form of a lariat. Group II splicing does not require any protein, or any external source of energy such as ATP or GTP. These lariat-RNA structures have adenosine as the "branch-point" residue, linked via a a 2'->5'   phosphodiester bond to a guanosine residue, and a 3'->5' phosphodiester bond to a pyrimidine residue. Studies on the specificity of the nucleobase requirement at the branch-accepting point have shown that the replacement of the central branch-accepting adenosine residue by any other nucleotide in mutants results in a considerable decrease in the efficiency of the splicing as compared to the wild type. Mutation experiments have shown that all four nucleotides can serve as the branch-acceptors, but it is adenosine which fulfills the structural criteria for the second step of splicing optimally, whereas cytidine residue is preferred to guanosine and uridine residues.
 

Figure 1. The two steps in Group II and Nuclear pre-mRNA Splicing. (Left) : Substrate (E1-IVS-E2), A = Branch-point Nucleotide. (Middle) : the 2'-OH of the branch-point adenosine (A) attacks the 5'-phosphate of G residue connecting the E1 exon giving the Lariat intron which is still covalently linked to the exon E2; (Right):  Upon formation of the Lariat intron, 3'-OH of E1 attacks the 5'-phosphate of E2 giving the ligated exons (E1 + E2) as products.. In Group II Splicing, no external source of energy such as ATP or protein required, only cofactor required is Mg2+ ion. Nuclear mRNA splicing reaction takes place within the spliceosome consisting of 50S to 60S complex of the pre-mRNA, four small ribonucleoprotein particles and as yet unknown number of associated protein factors, ATP and Mg2+ ion.

The above observations have prompted us to address to the following important questions regarding the mechanism of RNA splicing:

(1) Why is a lariat ubiquitously formed in RNA splicing reactions in eukaryotes?
(2) What are the unique structural features in the lariat that control the fidelity of the RNA splicing and, hence, protein biosynthesis in our cell?
(3) Since no ATP or GTP is required in the Group II splicing, and two covalent bonds are formed and broken, therefore the total energy input and output are balanced; these transesterification reactions should still need free energy of activation for covalent bond forming or -breaking reactions! Where does the free energy of activation come from in the transesterification reactions in the splicing? What is the energy inventory of the transesterification reactions involved in the splicing?
(4) Is there any structural and conformational basis for the evolutionary choice of adenosine as the branch-accepting nucleotide in the splicing reaction?
(5) How the conformation of the lariat is changed when the "branch-point" adenosine is replaced by cytidine, guanosine or uridine nucleotide?
(6) Does the chemospecific choice of adenosine nucleotide at the branch-point by Nature give the splicing reaction in vivo  a thermodynamic or a kinetic advantage that it can not have with any other nucleobase?

In order to answer these questions, we have developed synthetic methodologies to prepare model branched RNAs and lariat RNAs mimicking those of the branch-point of the natural lariat and have analyzed their tertiary structures by highfield isotope-edited NMR spectroscopy.

The summary of our results:

The conformation of the branched RNAs is dictated by the actual nucleobase composition. Thus, adenosine at the branch-point has a completely different tertiary structure than the counterparts with either guanosine, cytidine or uridine as the branch-point nucleotide. Second, the competing 2'->5' and 3'->5' stacking at the branch-point acts as an 'energy pump' since these two different stacked states have different free energies associated with their conformations. Thus a transition of the 2'->5' stacked conformation in a branch-RNA or lariat-RNA to 3'->5' stacked conformation releases a quantum of energy that can provide the free energy of activation for the transesterification reaction in the splicing [see our papers in the PDFs 121, 137, 145, 144, 150, 151, 153, 162, 163, 170, 183, 185, 195, 198, 202].

New chemical methodologies are being developed (both by solid and solution phase procedures) in my lab to synthesize some important model lariat-RNAs (Figure 2) that mimic that of the natural lariat-RNA of Fig. 1 more closely. Some of these efforts have resulted into the first unequivocal synthesis of lariat-RNAs [Figuire 2, see our papers in the PDFs 193, 206]. The most remarkable observation in the studies of these synthetic model lariat-RNAs is that the tetrameric and the pentameric loop in the lariat hexamer and heptamer self-cleaved to give corresponding branched RNA with a 5'-OH terminus and a 2',3'-cyclic phosphate, which is the hallmark of the first step of the catalytic self-cleavage reaction (the second being the catalytic turnover!) [see our papers in the PDFs 218, 225, 240].

    These synthetic lariat-RNAs are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k = ~1 min-1 at 37 °C), and their rates of the self-cleavage are also much slower (k = 0.25 x 10-4 min-1 for lariat hexameric lariat-RNA, and 0.16 x 10-3 min-1 for lariat heptamer at 22 °C).

    This slower self-cleavage rate of hexameric and hepatmeric lariat-RNAs, compared to the natural hammerhead have enabled us to perform their full conformational analysis by NMR spectroscopy [see our papers in the PDFs 218, 225, 240]. We have also shown that the trinucleotidyl loop in the tetrameric and pentameric lariat-RNAs [see our paper in the PDF 209] are completely stable.

    Our NMR studies have revealed the global as well as the local conformation of the self-cleavage site and the dynamics of the tetranucleotidyl and pentanucleotidyl loop in the hexameric or heptameric lariat-RNA [see our papers in the PDFs 218, 225], which has led us to understand, for the first time, the unique conformational requirements of the sugar-phosphate backbone in the lariat-RNAs that undergo the self-cleavage reaction akin to the natural ribozyme.

    In our search to explore the optimal structural requirement for the self-cleavage reaction of RNA, we have now synthesized a lariat-RNA analog in which the branch-point adenosine has a 2',5'-linked tetranucleotidyl loop and a 3'-ethylphosphate moiety mimicking the 3'-tail of the lariat-hexamer. What we found unique is that the 3'-ethylphosphate function at the branch-point in is the key structural feature that orchestrates its self-cleavage reaction (k = 0.15 x 10 -4 min-1 at 19 °C) to give acyclic branched RNA, compared to the stable 2',5'-linked cyclic RNA or 3',5'-linked cyclic RNA  [see our paper in the PDF 240]. We have subsequently worked out [PDF 240] the detailed conformational features of the self-cleaving tetrameric lariat-RNA by 600/800 MHz NMR spectroscopy and molecular dynamics simulations in the aqueous environment, which we are now in a position to use in the design of a suitable ribozyme.
 

Note, what is unique in the above work is that whereas Nature performs the chemospecific transesterification of a single phosphodiester bond out of a thousand or more by specific folding, we have achieved, for the first time in the lab, by design, a similar specific transesterification reaction in which one phosphodiester bond is specifically cleaved out of six others in the lariat heptamer (6) without the help of any cofactor, simply by the bending of the sugar-phosphate backbone.

Interestingly, our NMR work on the self-cleavage and geometry of the self-cleaving center is gradually giving us a very good understanding of the dynamic geometry of the bent sugar-phosphate backbone both at the self-cleavage site as well of the other constituent stable internucleotidyl phosphates.

Efforts are now underway to synthesize other lariat-RNAs that would have larger loop (hexa- to octadecanucleotidyl) till we have seen the self-cleavage rate arriving at the plateau. Note that in order to reach the goal for the synthesis of larger lariat loop, we are trying to come up with new effective synthetic methodologies such as stereospecific phosphorylation techniques, orthogonal protecting group strategy, intramolecular transesterification reaction etc. We also aim to determine the conformation of each of these self-cleaving lariat-RNAs by 600/800 MHz NMR spectroscopy and figure out the optimal tertiary structural requirement for the self-cleavage reactions. These studies should allow us to examine the local geometry of the self-cleaving lariat-RNAs within a family of lariat-RNAs and observe how does the conformational constrain of the lariat-loop dictate their unique self-cleavage reaction. Clearly, the understanding of the hyperspace of the geometry of the self-cleaving sites would open some new exciting possibilities also for the design of the ribozyme.

Design of Ribozyme Mimics Basing on the Self-Cleavage  of Lariat-RNA and Branched-RNA
 


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