LARP4B PAR-CLIP experiment. (A) In vivo crosslinking of LARP4B and RNA. HEK293 cells stably expressing Flag/HA-tagged LARP4B were cultured in 4SU containing medium. After crosslinking LARP4B was immunoprecipitated, copurified RNA was partially digested and radioactively labeled at the 5′ end. The LARP4B–RNA complex was resolved by SDS-PAGE and bound RNA detected by autoradiography (lane 1). For control, UV-treatment (lane 2) or 4SU (lane 3) was omitted. (B) T:C transition in PAR-CLIP reads demonstrate efficient crosslinking of LARP4B and RNA. Kept reads were aligned to the human genome and the identified mismatches are depicted. (C) LARP4B interacts preferentially with 3′ UTRs. Alignment of binding clusters showed that LARP4B mainly binds to exonic regions. Here, the association with coding sequences and 3′ UTRs is favored. (D) LARP4B binding is enriched toward the end of the mRNA. The PAR-CLIP signal strongly increases toward the end of the CDS whereas it is almost evenly distributed throughout the 3′ UTR. This leads to an increase in LARP4B binding toward the end of the mRNA. (E) The LARP4B PAR-CLIP is highly reproducible. The number of T:C transitions per gene correlates significantly between the two PAR-CLIP replicates.
