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

Ying Liu,Jinfeng Liu,Johnny J He,Zenyuan Wang

doi:10.1186/s13578-015-0032-z

Abstract

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 p110nrb, 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.

Highlights

RNA–protein interactions play important roles in gene expression control
Identification of Tip110 RNA ligands by the yeast three‐hybrid cloning To determine if Tip110 bound to specific RNAs, we took advantage of a RNA–protein interaction-based yeast three-hybrid cloning strategy [34]
The results showed that of these 156 colonies only 6 were no longer β-galactosidase-positive, while other 150 appeared to activate LacZ gene expression in the absence of Gal4 AD-Tip110

Summary

Introduction

RNA–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). There are five major snRNP U1, U2, U4, U5, and U6 in the spliceosomes containing small nuclear RNA (snRNA) U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, and U6 snRNA, respectively [6]. Those snRNA are transcribed in the nucleus and exported to the cytoplasm after acquiring a m7Gcap [1, 2]. The core snRNP are initially assembled in the cytoplasm to contain snRNA, survival motor neuron protein, Gem-associated proteins, and Sm proteins, followed

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