LARP4B associates with AU-rich sequences. (A) LARP4B-binding motif search. A binding motif search was performed as 7mer frequency count in 41-nt windows centered around the crosslinked centered region of the top 1000 binding clusters in 3′ UTRs. The 20 most abundant sequences are depicted. (B) Recombinant LARP4B expressed in E. coli was used for in vitro binding studies. (C) RNA sequences for in vitro binding studies. An RNA sequence of the CKB 3′ UTR was used as LARP4B target sequence. This sequence contains an U-rich region. To assay for binding of LARP4B to this stretch, a deletion mutant, CKB del, was used. A nontarget RNA sequence, TOP3B, was used to control for specificity. (D) LARP4B binds to target mRNA sequence. CKB RNA sequence was incubated with increasing amounts of recombinant LARP4B. The unbound and bound RNA was separated by native gel electrophoresis and detected by autoradiography, confirming binding of LARP4B to target RNA sequence. (E) Quantification of LARP4B binding to CKB RNA resulted in a dissociation constant (Kd) of ∼200 nM. (F) Using the CKB deletion mutant missing the U-rich stretch did not result in LARP4B binding, showing the dependency of AU-rich sequences for the interaction. (G) Electromobility shift assay with TOP3B RNA did not result in LARP4B binding confirming the specificity of LARP4B binding to target sequences.
