2016; 7:10615. 3ss and CPI-1205 branch points of a PUF60-dependent exon and the 3ss choice was also influenced by alternative splicing of on each intron in a step-wise manner, employing U1, U2 and U4/5/6 small nuclear ribonucleoprotein particles (snRNPs) and many non-snRNP proteins (1). A critical step in the spliceosome assembly is the recruitment of U1 snRNP to 5 splice sites (5ss) and U2 snRNP to the branch point (BP) (1), which is usually facilitated by binding of the U2 auxiliary factor (U2AF) to the 3 splice site (3ss) (2,3). U2AF is usually a stable heterodimer composed of the large subunit (U2AF65), which binds U-rich sequences in the pre-mRNA, including polypyrimidine tracts (PPTs) of most annotated 3ss (4), and the small subunit (U2AF35), which binds the 3ss AG dinucleotide through its zinc finger domains (5). However, U-rich sequences preferentially interact with a number of other RNA-binding proteins (RBPs), including hnRNP C (6), TIA-1/TIAR (7), SRSF3 (8), PTB (9) or PUF60 (10), often in a cooperative or competitive manner. How exactly their binding regulates exon inclusion in mature transcripts on a global scale remains poorly comprehended. PUF60 (poly-U-binding factor 60 kDa, also known as FIR, Hfp or Ro-bp1) is usually a splicing factor homologous to U2AF65 (10). PUF60 has two central RRMs and a C-terminal U2AF-homology motif (UHM), but lacks the N-terminal arginine/serine-rich (RS) and UHM ligand motif (ULM) domains present in U2AF65 (11,12) (Physique ?(Figure1A).1A). The PUF60-UHM does not bind nucleic acids (13) but interacts with tryptophan-containing ULMs in U2AF65, SF1 and SF3B1 (11). The PUF60-UHM and U2AF65-UHM have distinct binding preferences to ULMs at the N terminus of SF3B1 (11), a key U2 snRNP component that serves as a platform for UHM-containing spliceosome assembly factors (reviewed in 14). PUF60 activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA (10) and the relative abundance of PUF60 and U2AF65 can influence the choice of alternative splice sites (15). PUF60 and U2AF65 can bind SF3B1 ULMs simultaneously and noncompetitively (11), nevertheless RNA sequencing (RNA-Seq) studies revealed exons repressed by U2AF and activated by PUF60 (16), consistent with additional protein partners participating in the tight 3ss control. Apart from the role in splicing, anti-PUF60 antibodies co-precipitated RNA polymerase II C-terminal domain name and three components of the general transcription factor TFIIH, linking PUF60 to transcription (17). However, the exact function of PUF60 in global RNA processing has been unclear, despite the requirement of this protein for cell viability, proliferation and migration and a frequent overexpression in (pre-)malignant tissues (18,19). Open in a separate window Physique 1. RNA-Seq of HEK293 cells depleted of PUF60 CPI-1205 and RBM39.?(A) Domain name structure. (B) Western blot analysis of HEK293 cells lacking or overexpressing PUF60 CPI-1205 (homolog of RBM39 (rsd1) was found to bridge U1 and U2 snRNP contacts by binding the U1 snRNP core protein U1A and Prp5 ATPase, which interacted with the SF3B1 homolog (26). RBM39 and U2AF65 share the N-terminal RS Mmp13 domain name, which is usually absent in PUF60 (Physique ?(Figure1A),1A), and also RRM1/RRM2 and the C-terminal UHM (12). CPI-1205 The U2AF65-ULM binds the RBM39-UHM with a binding affinity over four orders of magnitude weaker than binding of the U2AF65-ULM to U2AF35-UHM (27C29), yet as much as 20% of alternatively spliced exons appear to be co-regulated by RBM39 and U2AF65 (30). Down-regulation of RBM39 decreased the expression of cell-cycle progression regulators (31), but RBM39 function in individual RNA processing actions remains poorly comprehended. Recently, independent studies have found heterozygous mutations in patients with a variable developmental delay, intellectual disability, spinal segmentation defects, and cardiac, ocular and renal abnormalities, first described for 8q24.3 microdeletions by Dauber (32C36). Apart from protein-truncating mutations seen in most patients, missense RRM/UHM variants totalled to a third of all reported cases (32C36), suggesting that the loss of PUF60 function in PD might be conferred also by impaired RRM and UHM interactions. However, the impact of these PD alleles on RNA processing has not been examined and the development of individual PD symptoms has not been understood. In this study, we have identified and characterized PUF60- and RBM39-dependent exons and examined functional consequences of mutations associated with human PD. MATERIALS.