Structure-guided U2AF ⁶⁵ variant improves recognition and splicing of a defective pre-mRNA

Abstract

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF 65 normally recognizes a Py tract consensus sequence preceding the major class of 3′ splice sites. We found that neurofibromatosisor retinitis pigmentosa-causing mutations in the 5′ regions of Py tracts severely reduce U2AF 65 affinity. Conversely, we identified a preferred binding site of U2AF 65 for purine substitutions in the 3′ regions of Py tracts. Based on a comparison of new U2AF 65 structures bound to either A-or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF 65 would specify U over other nucleotides. We found that the crystal structure of the U2AF 65 -D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF 65 affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF 65 binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF 65 variant is capable of manipulating gene expression in eukaryotic cells.pre-mRNA splicing | protein-RNA complex | protein engineering | crystal structure | RRM A pproximately 15% of the documented disease-causing point mutations disrupt consensus splice site elements in premRNAs, including a polypyrimidine (Py) tract between a branch point sequence (BPS) and an AG dinucleotide at the junction of the 3′ splice site (1) (Fig. 1A). For example, disease-causing mutations in Py tracts have been documented in ∼3,000 genes in the Human Gene Mutation Database (2), and an estimated 20% of these mutations affect regulatory splice site signals (3, 4). One of the earliest reports of a splice site mutation as a major cause of inherited human disease was for β-thalassemia (reviewed in ref. 5), for which splice site mutations in the human β-globin gene (HBB) are found in ∼14% of patients, causing symptoms of mild to severe anemia (reviewed in ref. 6).With the emergence of high-throughput sequencing technologies, splice site mutations in specific transcripts have been identified as common contributors to neuromuscular disorders, metabolic disorders, cancers, leukemias, deafness, and blindness, among other disorders (reviewed in ref. 4). Retinitis pigmentosa, the most prevalent form of inherited blindness in adults, represents one such disease that is primarily the consequence of mutations in splice sites of vision-relevant transcripts or splicing factors responsible for their recognition (reviewed in ref. 7). Neurofibromatosis type I, a disease characterized by tumors of nerve tissue, is an inherited disorder in which nearly 30% of the documented mutations disrupt neurofibromin 1 (NF1) splice sites (reviewed in ref. 8). Despite etiologic progress, the relationships between disease-causing pre-mRNA splice s...

An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal

“…The discovery of nine U2AF 65 -binding sites for contiguous Py-tract nucleotides was unexpected. Based on dU2AF 65 1,2 structures 14 15 20 , we originally hypothesized that the U2AF 65 RRMs would bind the minimal seven nucleotides observed in these structures. Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide ( Fig.…”

Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxide

“…To define the binding sites, the RNA/DNA‐bound structures of snRNP70 (PDB code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PKD, Kondo et al ., ), hnRNP I (PDB code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2AD9, Oberstrass et al ., ), and U2AF2 (PDB code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4TU8, Agrawal et al ., ) were aligned to the structure 4PKD using the structure alignment tool CE (Guda et al ., ) in order to identify similar structures. The binding sites were defined by residues situated ≤ 8 Å from the bound RNA/DNA.…”

“…To define the binding sites, the RNA/DNA-bound structures of snRNP70 (PDB code 4PKD, Kondo et al, 2015), hnRNP I (PDB code 2AD9, Oberstrass et al, 2005), and U2AF2 (PDB code 4TU8, Agrawal et al, 2014) were aligned to the structure 4PKD using the structure alignment tool CE (Guda et al, 2004) in order to identify similar structures. The binding sites were defined by residues situated ≤ 8 A from the bound RNA/DNA.…”

“…35 A when hnRNP I and U2AF2 aligned with snRNP70, respectively. Notably, these three proteins were cocrystalized with mRNA, and their aromatic amino acids (F213 and F171 in snRNP70; F98 and H62 in hnRNP I; and F304 and F262 in U2AF2) consistently formed strong vdW contacts with the rings of RNA (Agrawal et al, 2014;Kondo et al, 2015;Oberstrass et al, 2005) ( Fig. 1B).…”

A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

“…High resolution structures show the U2AF2 RRMs in an open, side-by-side arrangement when bound to strong Py tracts comprising contiguous uridines (28,29). The N-terminal U2AF2 RRM1 is more promiscuous than the uridine-stringent RRM2 (30,31). In the absence of RNA, a closed conformation masks the typical RNA binding surface of RRM1, leaving only RRM2 available (29).…”