Solution Structures of Nonameric and Decameric Branched- RNA Modelling the Lariat of Group II and Nuclear pre-mRNA Introns (Splicing) by 500 MHz NMR Spectroscopy

Conformational analysis of nonameric and decameric branched-RNAs by 500 MHz NMR spectroscopy (HOHAHA. DQFGOSY, NOESY, RORSY and temperature-depetuknt ckmical shifts and coupling cmstants) in conjunction wbh our previous studtes on trimers. tetramers. pentomcr and kptamer confinnr some general trends on tk conformational properties of branched RNA : (I) Tk corgormOion of tk sugar ring qf tk adenosine branch-point (Aq) is determined by tk presence or absence of a S-terminal nucleotide: Tk sugar qf tk A4 is in tk S-type conformation wkn no nucleotide is linhed to the S’-hydroxyl of A4 (as in trimer I and pentamer 3) while it is in tk N-type conformation wkn at least one nucleotide is linbd to its 5’hy&oxyl (as in tetramer 2, kptanter 4, nonamer 5 and decamer 6). (2) Tk 2*-linkd Gg is anti in tk trimer and pentamer. It is syn in tk tetramer, kptamw, nonamer and decanter. (3) Tk confomadon of tk kanckd trimer and pentamer is &nninated by a strong A4(2’-#)Gg kse-base staching. Tk A4(2’+S)Gg base staching is weakr in tk tetramw, heptamer. nonamer and decamer. (4) A wmparison qf tk tetramer, kptamer. nonamer and &canter shows that tk sugar cot&mation of tk nucleotides in tk S-chain (V3 in kpta, nona and decamer; V3 and C2 in decamer) are not infrucnced by tk introduction of additional pyrimidine nucleotides. (5) The enlargment of tk RNA branch system from tk 2’and 3’-termini leads however to some confomational differences amongst tk nucleotides at tk 2’and 3*-termird in tk branckd-RNAs possessing at least one S-terminal nucleotide (as in tetramer 2. kptamer 4. mummer 5. decamer 6): (a) Tk introductton of a 2’and 3’termbml A7 and GIO purine nucleotide sh&fts tk co@nnation of tk V9 and C6 sugarsfrom tk N-type in the pentamer and kptamer to tk S-type in the nonamer and decamer. (b) All tk nucleottdes of tk 2’and 3’ ckin have a S-type sugar. (c) Tk branch-point A4 which was in tk C3’-en& anti conformation in tk tetramer and kptamer is in tk Wendo, syn conformation in tk nonamer and decanter. Thus, three distinctly dfferent rvpcs of coafonnational features have ken identifiidjrom our strobes on branckd-RNA systems as models for lariat intron: Tkjirst group (trtmer I and pentamer 3) is characterized by A4 in a C2*-endo, syn conformation and a overall conformcrtio dominated by a strong A4(2’-W)Gg staching. The second group (tetramer 2 and kptamer 4) is character&d by A4 in a W-endo, anti conforwmtion and a weaker Ag(2’+5>Gg staching. Tk third group (nonamer 5 and decamer 6) is charactetised by C3*en&, syn conformadon for tk branch-point A4 residue, wsakrA4(2’+5’)Gg staching and tk nuckotidcs of the 2’and J’-chains are all in tk CZ’-endo conformation which indicates that the 2’and 3’-chains in branckd nonamer 5 and decamer 6 do not adopt an A-RNA type helix. In the Group II and nuclear mFCNA splicing, the intton is excised as a 29’ branched circular RNA called Lariat’“. The lariat is fortned, at the penultimate step of the ligation of two exons, by the nucleophilic attack of the 2’-hydroxyl of the branch-point adenosine nucleotide, located near the 3’-end of the intron, on the Sphosphate of a guanosine nucleotide located at the S-end of the intron (Figure 1). The branch-point adenosine nucleotide in the Lariat thus carries a 2’+5’ phosphodiester linkage in addition to the nortnal 3’+5’ phosphodiester linkages (Figure 1). The nucleotide sequence at the branch-site is highly conservebd. It is always an adenosine nucleotide that constitutes the branch-point. The 2’-+5’ linked nucleotide is invariably a guanosine residue. The 3’-hydroxyl of the branch-point adenosine is 3’+5’ phosphodiester linked to a pyrimidine nucleotide, and its S-hydroxyl is phosphodiester linked to a uridine or adenosine (see Figure 1). It