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Abstract
In yeast, the U2 small nuclear ribonucleic acid (snRNA) component of the spliceosome
is targeted for additional post-transcriptional modifications in response to cellular
stress. Uridines 56 and 93 are both modified to pseudouridines (Ψ) during nutrient
deprivation, while U56 is also pseudouridylated during heat shock. Both positions
are located within stem II, which must toggle between two mutually exclusive structures
during splicing. Stem IIa forms during spliceosome assembly, and stem IIc forms during
the catalytic steps. We have studied how uridine 56 and 93 pseudouridylation impacts
conformational switching of stem II. Using single-molecule Förster resonance energy
transfer, we show that Ψ56 dampens conformational dynamics of stem II and stabilizes
stem IIc. In contrast, Ψ93 increases dynamics of non-stem IIc conformations. Pseudouridylation
impacts conformational switching of stem II by Mg2+ or the U2 protein Cus2; however,
when Mg2+ and Cus2 are used in combination, the impacts of pseudouridylation can be
suppressed. These results show that stress-induced post-transcriptional modification
of U56 and U93 alters snRNA conformational dynamics by distinct mechanisms and that
protein and metal cofactors of the spliceosome alter how snRNAs respond to these modifications.