SUMMARY Mutations affecting spliceosomal proteins are the most common class of mutations in patients with myelodysplastic syndromes (MDS), yet their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2’s normal sequence-specific RNA binding activity, thereby altering recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2 that triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in splicing of key regulators, and impaired hematopoiesis.

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 39 splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 39 splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allelespecific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 39 splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 39 splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allelespecific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3 ′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3 ′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5 ′ and 3 ′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.

Substantial effort is currently devoted to identifying cancer-associated alterations using genomics technologies. While the genome is inherently stable over short time frames, the transcriptome is dynamic and potentially susceptible to alteration as samples move from the patient to the lab bench. Here, we show that standard blood collection procedures rapidly change the transcriptional and post-transcriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways, up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms, and inhibition of RNA surveillance. These artifacts affect almost all published leukemia genomics studies, explaining up to 40% of putative cancer-associated differential expression and alternative splicing. To determine how standard blood collection procedures affect hematopoietic transcriptomes, we collected whole blood from four healthy donors in anticoagulant blood collection tubes. We then left this blood at room temperature for defined lengths of time (0-48h) in order to mimic the variable incubation that patient samples are typically subjected to during transfer from the primary treating physician to a research center, isolated peripheral blood mononuclear cells (PBMCs), and performed deep RNA-seq (Figure A). Contrary to the common assumption that RNA degrades during incubation, we observed no evidence of decreased RNA quality. Nonetheless, rapid and dramatic changes affected virtually every level of the gene expression process. The changes were highly biased; for example, pseudogenes and antisense RNAs were preferentially up-regulated, while cassette exons were preferentially repressed (Figure B). Incubation-induced changes in the transcriptome confound the identification of true cancer-specific alterations. Many genes affected by incubation participate in biological pathways of current interest in leukemia, including cytokine production, NF-κB signaling, chromatin modification, and RNA splicing. Furthermore, incubation for as little as 4 hours, our shortest time point, introduced dramatic changes in the post-transcriptional landscape. We observed widespread isoform switches, wherein isoforms that were rare or even undetectable at 0h became the major isoform after 8 to 24h of incubation, in genes such as NOTCH2, LEF1, and PHF20 that have been previously used as leukemic biomarkers (Figure C). Perhaps most surprisingly, incubation rapidly inhibited RNA surveillance, leading to genome-wide expression of normally degraded RNAs. This highly abnormal RNA surveillance inhibition was readily detectable in all published leukemia genomics datasets that we analyzed, with the exception of TCGA, and undetectable in any lymphoma or solid tumor dataset (Figure D). Together, our data show that incubation-induced changes in the transcriptional and post-transcriptional landscapes generate substantial artifacts that complicate the interpretation of leukemia genomics studies. To facilitate the incorporation of sample processing artifacts into downstream analysis, we provide biomarkers that detect prolonged incubation of individual samples. We furthermore show that the simple expedient of keeping blood on ice drastically reduces changes to the transcriptome. Our findings have important implications for the interpretation of published and ongoing leukemia genomics studies. Figure. (A) Leukemic samples are frequently subject to an ex vivo incubation period of variable length. (B) Ex vivo incubation causes biased up-regulation of pseudogenes and antisense RNAs and repression of cassette exons. (C) Incubation causes complete isoform switches in the LEF1 and PHF20 genes. (D) RNA surveillance is inhibited after only 4h of incubation. This abnormal inhibition of RNA surveillance is visible in all analyzed leukemia genomics datasets, with the exception of TCGA, and not in any lymphoma or solid tumor. Numbers above each x axis label indicate the number of samples in each dataset. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.