ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways [iCLIP-seq]

we try to investigate the binding of ALYREF and NXF1 on histone mRNA when the cell treated with indicated siRNAs. ALYREF plays key roles in nuclear export of polyadenylated mRNAs and also modulates their 3' processing, but whether it is involved in regulating RNAs beyond polyadenylated mRNAs is unknown. The replication-dependent (RD) histone mRNAs are not polyadenylated, but end in a stem-loop (SL) structure. Here we demonstrate that ALYREF prevalently binds a region next to the SL on RD histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and ensures this binding. To examine how SLBP KD impact ALYREF distribution on the histone mRNA, we carried out ALYREF iCLIP in control and SLBP KD cells. To investigate the functional consequence for ALYREF binding on histone mRNAs, we isolated polyA+ and polyA- RNAs from control and ALYREF KD cells, and carried out RNA-seq separately. The ALYREF-iCLIP assay was performed in control and SLBP knock down cells using ALYREF antibody. The NXF1-iCLIP experiments were carried out in Flag-NXF1 stable expressed cells treated with control, ALYREF and SLBP siRNAs for 72 hr using Flag antibody.