A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation.

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

A Broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF(65).

U2AF(65) is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF(65) recognize a polypyrimidine tract at the 3' splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF(65) RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF(65) conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF(65) conformational pool.

Formation of the RFX gene regulatory complex induces folding of the interaction domain of RFXAP.

Major histocompatibility complex class II (MHCII) molecules have a central role in the mammalian adaptive immune response against infection. The level of the immune response is directly related to the concentration of MHCII molecules in the cell, which have a central role in initiating the immune response. MHCII molecules are therefore a potential target for the development of immunosuppressant drugs for the treatment of organ transplant rejection and autoimmune disease. The expression of MHCII molecules is regulated by a cell specific multiprotein complex. The RFX complex is the key DNA binding component of this complex. The RFX complex is composed of three proteins-RFX5, RFXAP, and RFXB-all of which are required for activation of expression of the MHCII genes. Little is currently known about the precise regions of the RFX proteins that are required for complex formation, or their structure. We have therefore identified the key regions of RFX5, RFXAP, and RFXB, which are required to form the RFX complex and have characterized the individual domains and the complexes they form using NMR and circular dichroism spectroscopy and isothermal titration calorimetry. Our results support a model for the assembly of the RFX complex in which the interaction between RFX5 and RFXAP promote folding of a poorly structured region ofRFXAP, which is required for high affinity binding of RFXB to the RFX5.RFXAP complex.

Solution structure of the heterotrimeric complex between the interaction domains of RFX5 and RFXAP from the RFX gene regulatory complex.

The mammalian immune response is mediated by a heterotetrameric transcriptional control complex, called regulatory factor X (RFX), that regulates the expression of major histocompatibility complex class II genes. RFX comprises three proteins: RFX5 (two copies), RFXAP, and RFXB, and mutations and deletions that prevent the assembly of the RFX complex have been linked to a severe immunodeficiency disorder. Two RFX5 molecules and one RFXAP molecule assemble in the cytoplasm prior to nuclear localization, a process mediated by an N-terminal "dimerization domain" of RFX5 (RFX5(N)) and a C-terminal domain of RFXAP (RFXAP(C)). We previously presented evidence that RFXAP(C) is unstructured in the absence of RFX5(N) but adopts a regular structure in the RFX5(N)(2)-RFXAP(C) complex and that the RFX5(N)(2)-RFXAP(C) complex binds RFXB with high affinity. We now report the structure of the RFX5(N)(2)-RFXAP(C) complex, determined in solution by (15)N- and (13)C-edited NMR spectroscopy. RFX5(N) consists of a long central helix flanked by two shorter helices. The central helices of the two RFX5(N) molecules form an antiparallel coiled coil, and the flanking helices pack at the ends of the long helices in a perpendicular arrangement such that the RFX5(N) dimer is shaped like a staple. RFXAP(C) consists of two α-helices that form a V-shaped structure that packs within the RFX5(N)(2) staple. Leucine residues in the leucine-rich region of RFX5(N) (62-LYLYLQL-68) that are critical for major histocompatibility complex class II gene expression in vivo contribute to both the dimer (Leu64 and Leu68) and the RFX5(N)-RFXAP(C) interfaces (Leu62 and Leu66). The clustering of hydrophobic residues from different regions of RFXAP(C) suggests a potential binding site for RFXB.