The RNA Binding Motif Protein 15B (RBM15B/OTT3) Is a Functional Competitor of Serine-Arginine (SR) Proteins and Antagonizes the Positive Effect of the CDK11p110-Cyclin L2α Complex on Splicing

Pascal Loyer,Adeline Busson,Janeen H Trembley,Judith Hyle,Jose Grenet,Wei Zhao,Catherine Ribault,Tristan Montier,Vincent J Kidd,Jill M Lahti

doi:10.1074/jbc.m110.192518

Abstract

Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.

Highlights

The human cell division control 2-like genes Cdc2L1 and Cdc2L2 encode two related protein kinases, denoted CDK11B and -A, respectively, which are expressed as two predominant protein isoforms designated by their apparent molecular mass (p110 and p58 for the 110- and 58-kDa isoforms, respectively)
In addition to RNPS1, a known interactor of CDK11p110 [10], one of the proteins we identified in this screen was a partial cDNA that encoded the majority of the RNA binding motif 15B protein (RBM15B/ OTT3) but lacked the 5Ј-end of the full-length cDNA
RBM15B is highly similar to RBM15/OTT1, which was originally discovered as a fusion partner with the gene megakaryoblastic leukemia 1 (MKL1) in the translocation t(1;22)(p13;q13) associated with childhood acute megakaryocytic leukemia [28, 29]

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screen—The yeast two-hybrid screen was performed as described previously [10] using the full-length CDK11Bp110 (Cdc2L1) fused to the GAL4 DNA binding domain and cloned into the PAS1CYH2 plasmid and a human B-cell cDNA library subcloned into the pACT plasmid containing the GAL4 activation domain Transfections of HEK293T cells with expression vectors encoding CDK11p110, cyclins L1␣ and L2␣, 9G8, SF2/ASF, and various RBM15B constructs were performed using the transfection reagents FuGENE 6 (Roche Applied Science), JetPEI (Qbiogene), or KLN47 [23], and cells were harvested 48 h after transfection. GST Pulldown Assay—Pulldown assays were performed by incubating GST-RBM15B fusion proteins (supplemental Data S5) with [35S]methionine-labeled in vitro transcribed and translated (IVTT) products or total lysates of either nontransfected HEK293T to detect endogenous CDK11p110 and SR proteins or transiently transfected HEK293T to detect cyclins L1␣ and L2␣, SF2/ASF, and 9G8. The optimal amounts of DNA expression vectors for RBM15B, CDK11, cyclin L2␣, SF2/ASF, and 9G8 used in the transfections were established in pilot experiments that were performed to optimize protein expression (data not shown). Immunoblot analyses of equal volume cell lysates were performed using anti-CDK11, -cyclin L2␣, -RBM15B, -9G8, and -SF2/ASF polyclonal antibodies. Jude Children’s Research Hospital, Memphis, TN) have developed a specific non-parametric rank-based test as described previously [8] that is available upon request

RESULTS

To confirm this hypothesis and to determine whether

DISCUSSION