Tip110 binding to U6 small nuclear RNA and its participation in pre-mRNA splicing.

backgroundRNA-protein interactions play important roles in gene expression control. These interactions are mediated by several recurring RNA-binding motifs including a well-known and characterized ribonucleoprotein motif or so-called RNA recognition motif (RRM).

resultsIn the current study, we set out to identify the RNA ligand(s) of a RRM-containing protein Tip110, also known as p110(nrb), SART3, or p110, using a RNA-based yeast three-hybrid cloning strategy. Six putative RNA targets were isolated and found to contain a consensus sequence that was identical to nucleotides 34-46 of U6 small nuclear RNA. Tip110 binding to U6 was confirmed to be specific and RRM-dependent in an electrophoretic mobility shift assay. Both in vitro pre-mRNA splicing assay and in vivo splicing-dependent reporter gene assay showed that the pre-mRNA splicing was correlated with Tip110 expression. Moreover, Tip110 was found in the spliceosomes containing pre-spliced pre-mRNA and spliced mRNA products. Nonetheless, the RRM-deleted mutant (ΔRRM) that did not bind to U6 showed promotion in vitro pre-mRNA splicing, whereas the nuclear localization signal (NLS)-deleted mutant ΔNLS that bound to U6 promoted the pre-mRNA splicing both in vitro and in vivo. Lastly, RNA-Seq analysis confirmed that Tip110 regulated a number of gene pre-mRNA splicing including several splicing factors.

conclusionsTaken together, these results demonstrate that Tip110 is directly involved in constitutive eukaryotic pre-mRNA splicing, likely through its binding to U6 and regulation of other splicing factors, and provide further evidence to support the global roles of Tip110 in regulation of host gene expression.