A Broad Range of Conformations Contribute to the Solution Ensemble of the Essential Splicing Factor U2AF⁶⁵

U2AF⁶⁵ is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF⁶⁵ recognize a polypyrimidine tract at the 3′ splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF⁶⁵ RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF⁶⁵ 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 U2AF⁶⁵ conformational pool

Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs

Acquired point mutations of pre-mRNA splicing factors recur among cancers, leukemias, and related neoplasms. Several studies have established that somatic mutations of a U2AF1 subunit, which normally recognizes 3′ splice site junctions, recur among myelodysplastic syndromes. The U2AF2 splicing factor recognizes polypyrimidine signals that precede most 3′ splice sites as a heterodimer with U2AF1. In contrast with those of the well-studied U2AF1 subunit, descriptions of cancer-relevant U2AF2 mutations and their structural relationships are lacking. Here, we survey databases of cancer-associated mutations and identify recurring missense mutations in the U2AF2 gene. We determine ultra-high-resolution structures of the U2AF2 RNA recognition motifs (RRM1 and RRM2) at 1.1 Å resolution and map the structural locations of the mutated U2AF2 residues. Comparison with prior, lower-resolution structures of the tandem U2AF2 RRMs in the RNA-bound and apo states reveals clusters of cancer-associated mutations at the U2AF2 RRM–RNA or apo-RRM1–RRM2 interfaces. Although the role of U2AF2 mutations in malignant transformation remains uncertain, our results show that cancer-associated mutations correlate with functionally important surfaces of the U2AF2 splicing factor

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

<div><p>We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that <i>U2AF1</i>S34F is expressed from one of the two endogenous <i>U2AF1</i> loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential <i>in vitro</i> RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing <i>U2AF1</i> mutations do not require the mutant protein for growth <i>in vitro</i> or <i>in vivo</i>. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the <i>U2AF1</i>S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in <i>U2AF1</i>-mutant cells and why tumors harboring <i>U2AF1</i> mutations always retain an expressed copy of the wild-type allele.</p></div

Creation of isogenic lung cell lines that recapitulate features of S34F-associated splicing.

<p><b>(A)</b>. Strategy to create a T<u>C</u>T to T<u>T</u>T point mutation (S34F) at the endogenous <i>U2AF1</i> locus in HBEC3kt cells. TALEN, transcription activator-like effector nuclease; E, exon; mE, mutant (S34F) exon; ITR, inverted terminal repeat; HA, homology arm. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#sec002" target="_blank">Results</a> and Supplemental Materials and Methods for details. <b>(B)</b>. MUT1a and MUT1b cells contain similar levels of mutant and wild-type <i>U2AF1</i> mRNA. The number of reads supporting mutant or wild-type <i>U2AF1</i> was obtained from RNA-seq, using poly(A)-selected RNA from the four cell lines. <b>(C).</b> The S34F-associated cassette exons in <i>ASUN</i> and <i>STRAP</i> mRNAs show decreased inclusion in MUT cell lines. (Top) Scheme of alternative splicing with a cassette exon (black box) to generate short and long isoforms in which the cassette exon is excluded or included. (Bottom) Alternative splicing of cassette exons in <i>ASUN</i> and <i>STRAP</i> mRNAs, measured by RT-qPCR using isoform-specific primers. The short/long isoform ratio in WT1 cells was arbitrarily set to 1 for comparison. Asterisks represent statistical significant changes as compared to that in WT1 cells. Error bars represent s.e.m. (standard error of the mean) (n = 4). <b>(D)</b>. Heat map depicting the inclusion levels of all cassette exons that showed at least a 10% change in use among the cell lines. The dendrogram was constructed from an unsupervised cluster analysis. <b>(E).</b> Sequence logos from 3′ splice sites preceding cassette exons with altered inclusion in MUT1a cells display typical S34F consensus 3′ splice sites. Logos were constructed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#pgen.1006384.g001" target="_blank">Fig 1A</a> based on the transcriptome of MUT1a cells in comparison with that of WT1 cells. Other comparisons of transcriptomes from MUT and WT cell lines yielded similar sequence logos.</p

S34F-associated splicing program correlates with S34F:WT mRNA ratios in LUAD.

<p><b>(A)</b>. Different consensus 3′ splice site sequences preceding cassette exons for two representative LUADs with the S34F mutation computed from published TCGA transcriptomes. In both cases, changes in the use of cassette exons were determined by comparisons with an average transcriptome from LUADs without <i>U2AF1</i> mutations. Boxes highlight nucleotides found preferentially at the -3 position. The nucleotide frequencies preceding exons that are more often included or more often excluded in tumors with the S34F mutation differ from the genomic consensus in the tumor from patient #7903, which represent “typical S34F” consensus 3′ splice sites, but not in the tumor from patient #7727, which represent “quasi-WT” consensus 3′ splice sites. (Consensus 3′ splice sites from transcriptomes of every S34F-mutant LUAD are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#pgen.1006384.s002" target="_blank">S1 Fig</a>). The analysis was restricted to introns with canonical GT-AG U2-type splice sites. The invariable AG at the 3′ splice site was not plotted to scale in order to highlight the consensus sequences at the -3 position. The vertical axis represents the information content in bits (the maximal value is two. Zero to one bit is shown). n is the number of cassette exon sequences used to construct the logo. A cartoon illustrating alternative splicing of a cassette exon (black box) is shown on the left side of the sequence logo. Black lines over splice junctions illustrate the S34F-promoted isoform for each comparison. <b>(B)</b>. <i>U2AF1</i>-mutant LUAD transcriptomes harboring “typical S34F” consensus 3′ splice sites have relatively high S34F:WT mRNA ratios. <i>U2AF1</i>-mutant LUAD samples were grouped based on the nature of the consensus 3′ splice sites. The asterisk represents a statistically significant change by Student’s t test. <b>(C)</b>. S34F:WT <i>U2AF1</i> mRNA ratios do not correlate with tumor purity in LUAD tumors with the S34F mutation. Tumor purity is represented by the percent of tumor nuclei in each LUAD sample (derived from TCGA clinical data) and plotted against the S34F:WT mRNA ratios. <b>(D)</b>. Inclusion of the <i>STRAP</i> cassette exon correlates with the S34F:WT mRNA ratio. Same as Panel <b>C</b> but the S34F:WT mRNA ratio is plotted against the inclusion frequency for the <i>STRAP</i> cassette exon. The median inclusion level of the same cassette exon for all transcriptomes from tumors without a <i>U2AF1</i> mutation (S34F:WT mRNA ratio = zero) is shown as a triangle. r, Pearson’s correlation coefficient. In panels B–D, circles represent samples with typical-S34F consensus 3′ splice site sequences; squares represent samples with quasi-WT consensus 3′ splice site sequences. Colors indicate <i>U2AF1</i> copy number status as calculated by GISTIC 2.0 (See details in the Supplemental Materials and Methods): black, diploid; blue, shallow deletion; red, gain.</p

The ratio of S34F:WT U2AF1 gene products controls S34F-associated splicing in isogenic HBEC3kt cell lines.

<p><b>(A)</b>. Reduction of both mutant and wild-type U2AF1 RNA and protein does not affect S34F-associated splicing. WT1 and MUT1a cells were transduced with shRNAs against <i>U2AF1</i> (shU2AF1#1 and #4) or two control shRNAs (shScbr, a scrambled shRNA; shGFP, an shRNA against <i>GFP</i>). Total RNA and protein were harvested 4 days later. The frequencies of incorporation of cassette exon sequences in <i>ASUN</i> and <i>STRAP</i> mRNAs (top and middle panels) were determined by the relative short/long isoform ratios by RT-qPCR, as represented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#pgen.1006384.g002" target="_blank">Fig 2C</a>. Immunoblots for U2AF1 and ACTB in total cell lysates are shown in the bottom panel. Asterisks represent statistical significant changes as compared to shScbr-transduced condition in each cell line. Error bars represent s.e.m (n = 3). <b>(B)</b>. Overexpression of wild-type or mutant <i>U2AF1</i> to change S34F:WT ratios alters S34F-sensitive splicing. WT1 and MUT1a cells were transduced with expression vectors encoding <i>GFP</i>, wild-type (WT) or mutant (S34F) <i>U2AF1</i> for 3 days before harvesting cells to quantify the level of splicing changes and proteins as in panel A. Asterisks represent statistical significant changes as compared to GFP-transduced condition in each cell line. Error bars represent s.e.m (n = 3). <b>(C)</b>. Disruption of WT <i>U2AF1</i> by gene editing to increase S34F:WT ratios enhances S34F-sensitive splicing. WT1 and MUT1a cells were transduced with Cas9 and either sgRNA-GFP or sgRNA-WT. Total RNA and protein were harvested 6 days later for assays as in panel <b>A</b>. Asterisks represent statistical significant changes as compared to Cas9 and sgRNA-GFP-transduced condition in each cell line. Error bars represent s.e.m. (n = 3).</p

Wild-type but not mutant <i>U2AF1</i> is required for the clonogenic growth of the isogenic HBEC3kt cells and LUAD cell lines.

<p><b>(A)</b>. Clonogenic growth assays after selective disruption of the wild-type or mutant <i>U2AF1</i> allele. Left panel: The indicated cell lines were transduced with lentiviruses expressing Cas9 and sgRNA-GFP, sgRNA-S34F or sgRNA-WT, followed by clonogenic assays. Cell colonies were stained with methylene blue and counted three weeks later. Right panel: Quantification of the clonogenic assay. The results are shown as percent clonogenicity by setting the number of control cell colonies (cells transduced with Cas9 and sgRNA-GFP) as 100%. Asterisks represent statistical significant changes as compared to Cas9 and sgRNA-GFP-transduced condition in each cell line. Error bars represent s.e.m (n = 3). <b>(B)</b>. Rescue of growth inhibition by Cas9 and sgRNA-WT by overexpressing a form of wild-type <i>U2AF1</i> cDNA that is not predicted to be the target for sgRNA-WT (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#pgen.1006384.s009" target="_blank">S8 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006384#pgen.1006384.s001" target="_blank">S1 Text</a>). Left panel: A549 cells were transduced with a control (DsRed-Express 2) or a wild-type <i>U2AF1</i> cDNA that is not predicted to be the target for sgRNA-WT. Increased expression of wild-type U2AF1 was confirmed by immunoblot (left bottom panel). These cells were subsequently transduced with Cas9 and either sgRNA-GFP or sgRNA-WT followed by clonogenic assays as in panel A (left upper panel). Right panel: Quantification as in Panel A. The asterisk represents a statistical significant change for the indicated comparison. Error bars represent s.e.m. (n = 3).</p