A new regiospecific synthesis of “Branched” tetraribonucleotide and its three analogues to delineate the chemospecific role of the “Branch-point” adenine nucleotide in splicing
✍ Scribed by N. Balgobin; A. Földesi; G. Remand; J. Chattopadhyaya
- Publisher
- Elsevier Science
- Year
- 1988
- Tongue
- French
- Weight
- 787 KB
- Volume
- 44
- Category
- Article
- ISSN
- 0040-4020
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✦ Synopsis
Aksbrtct The synthefes @four "branched" te~~o~leo~s 32 -35 ore reported using the key trimeric building blocks 19 -22 which have been convenient& prepared by the reactions of 18 with 14 -17 urin the ~t~logies developed in the ~-p~sp~~te chemistry. An appropriate choice o f the complementary 2 '-OH protecting groups in the intennediares 19 -22 has pennined a regioselective removal of one 2,-U~ protecdn~ groups (~-p~~~n-~-yi-), by a mild acid treatment, at the branch-accepting sugar moiety to give the inretmedia&s 23 -26 in high yields. These intermediates 23 -26 have been s~seq~~~ converted to the "branched" tetraribonucleotides 28 -31 by reactions with appropriateiy protected 5 '-phosphiteamidite block 27 in the M.Q& marmer. The ~ro~ctected *bran&e@ te~~~~eo~s 32 -35 have been subsequently characterized by IH-& 3IP-NMR spectroscopy. Two dimension& IH f3lP correhztion spectrosc5py of tkse branched te~~'~~ieo~s have ~eq~.vocQl~ ~t~blis~d the regiospecific sites of 3 * 3 5 *and 2 ' -4 5 ' phrsphodiester linkages. The removal of the introns from the pm-mRNA and ligation of tbc 3'-and 5'-coding regions (exons) to form a functions RNA for protein biosynthesis is generally called splicing l-5. In Group II and nuclear mRNA splicing, first, the pre-mRNA is cleaved at the 5'-splice site and a branched (lariat) intermed&e is formed. Then, in the second step, cleavage at the 3'-splice site and ligation of two exons leads to the release of the lariat intron l-1 1. These lariat structures have adenosine as the "branchpoint" residue, linked via a 2' + 5' phophodiester bond to a byline residue, and a 3' + 5'ph~ph~iester bond to a pyrimidine residueI-11. Specific nuckobase requirement of the branch-accepting point for the splicing in eukaryotes have bean recently reportedl2-14. Replacement of the central branch-accepting adenosine residue by any other nucleotide in mutants results in a considerable decrease in efftciency in the splicing as compared to the wild type. Mutation experiments have shown that all four nuckoti&s can serve as branch-acceptors, but it is adencaine and cytidine residues which are prefered to guanosine and uridinc residuesl*-13. Only branches to adenosine or cytidine participate effkiently in the second step of splicing in mutation experiments in vitro but the natural splicing in eukaryotes in vivo exclusively ptcfers adenosinc as the branch-point for error-free processing of pm-mRNA. We wished to study the structural basis for the evolutionary choice of adenosinc as the branch-accepting nucleotide in the splicing reaction by the confo~ation~ analysis of the model branched RNA lariats with substituted nucleotides (cytidine, guanosine and uridine) at the branch-point instead of adenosine. We chose a "branched" tetmribonucleotide molecuie, such as 32, as a representative of the biologically occurring lariat structure because it has been shown by II+-NMR spectroscopy that a simple branched ~~nucl~tide such as t has an unnatural ~nfo~a~on3~40~42