No abstract

Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles. Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome. We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing. Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site. We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6. Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.

No abstract

Glucocorticoids remain the most widely used immunosuppressive and anti-inflammatory drugs, yet substantial gaps exist in our understanding of glucocorticoid-mediated immunoregulation. To address this, we generated a pathway-level map of the transcriptional effects of glucocorticoids on nine primary human cell types. This analysis revealed that the response to glucocorticoids is highly cell type dependent, in terms of the individual genes and pathways affected, as well as the magnitude and direction of transcriptional regulation. Based on these data and given their importance in autoimmunity, we conducted functional studies with B cells. We found that glucocorticoids impair upstream B cell receptor and Toll-like receptor 7 signaling, reduce transcriptional output from the three immunoglobulin loci, and promote significant up-regulation of the genes encoding the immunomodulatory cytokine IL-10 and the terminal-differentiation factor BLIMP-1. These findings provide new mechanistic understanding of glucocorticoid action and emphasize the multifactorial, cell-specific effects of these drugs, with potential implications for designing more selective immunoregulatory therapies.