Abstract
U6 RNA contains a 1 × 2-nt internal loop that folds and unfold during spliceosomal assembly and activation. The 1 × 2 loop consists of a C67•A79 base pair that forms an additional hydrogen bond upon protonation, C67•A+79, and uracil (U80) that coordinates the catalytically essential magnesium ions. We designed a series of RNA and DNA constructs with a 1 × 2 loop sequence contained in the ISL, and its modifications, to measure the thermodynamic effects of protonation and magnesium binding using UV-visible thermal denaturation experiments. We show that the wild-type RNA construct gains 0.43 kcal/mol in 1 M KCl upon lowering the pH from 7.5 to 5.5; the presence of magnesium ions increases its stability by 2.17 kcal/mol at pH 7.5 over 1 M KCl. Modifications of the helix closing base pairs from C–G to U•G causes a loss in protonation-dependent stability and a decrease in stability in the presence of magnesium ions, especially in the C68U construct. A79G single-nucleotide bulge loop construct showed the largest gain in stability in the presence of magnesium ions. The DNA wild-type construct shows a smaller effect on stability upon lowering the pH and in the presence of magnesium ions, highlighting differences in RNA and DNA structures. A U6 RNA 1 × 2 loop sequence is rare in the databases examined.
Highlights
Eukaryotic translation of proteins requires excision of introns from pre-mRNA to form mature messenger RNA
Our experiments show that an additional hydrogen bond in the C67A+79 in the U6 internal stem–loop (ISL) adds 0.43 kcal/mol to RNA stability in 1 M KCl at pH 5.5
Neither C66U or C68U show an increase in stability in 1 M KCl in pH 5.5 over pH 7.5, with C66U showing 1.99 kcal/mol and C68U showing 2.39 kcal/mol lowered stability than wild-type construct in pH 5.5
Summary
Eukaryotic translation of proteins requires excision of introns from pre-mRNA to form mature messenger RNA. The spliceosome is a multimegadalton complex that functions as a ribozyme to excise introns and ligate exons in the process of creating mature messenger RNA (Fica et al 2013). It is comprised of more than 70 structural proteins as well as five catalytic small nuclear RNAs (snRNAs) designated U1, U2, U4, U5, and U6 (Rappsilber et al 2002; Zhou et al 2002). The U6 snRNA must undergo a series of structural rearrangements to assemble and activate the spliceosome and catalyze RNA splicing (Fortner et al 1993). The U2/U6 complex catalyzes splicing via two transesterification reactions (Datta and Weiner 1991; Madhani and Guthrie 1992; Valadkhan and Manley 2003)
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