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Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules
Published on: May 12, 2017
Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies
Giulia Biancon1, Poorval Joshi1, Joshua T Zimmer2
1Section of Hematology, Department of Internal Medicine, Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA.
Abstract:
Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.
Insights
Mutations in the U2AF1 splicing factor alter its RNA binding, leading to splicing errors and promoting myeloid cancers. These U2AF1 mutations also induce stress granule formation, a key adaptive cancer strategy.
Area of Science:
- Molecular Biology
- Cancer Genomics
- RNA Splicing Mechanisms
Background:
- Splicing factor mutations, particularly in U2AF1, are increasingly recognized as drivers of myeloid malignancies.
- The U2AF1/U2AF2 heterodimer plays a crucial role in defining the 3' splice site (3'SS), but the precise functional consequences of U2AF1 mutations on splicing are not fully understood.
Purpose of the Study:
- To elucidate how U2AF1 hotspot mutations affect its RNA binding and splicing functions in vivo.
- To investigate the downstream consequences of altered U2AF1 function on cellular processes, including stress granule formation.
Main Methods:
- Development of a crosslinking and immunoprecipitation (CLIP) procedure for single-nucleotide resolution analysis of U2AF1-RNA interactions.
- Integration of RNA binding, splicing, and RNA turnover data.
- Single-cell RNA sequencing (scRNA-seq) to assess cellular responses.
Main Results:
- U2AF1 S34F and Q157R mutants exhibit altered contacts with the 3'SS AG sequence at specific nucleotide positions (-3 and +1, respectively).
- These mutations disrupt U2AF2-RNA interactions, leading to aberrant splicing events such as intron retention and exon skipping.
- U2AF1 mutations were predicted to affect stress granule components, and this was experimentally confirmed in U2AF1-mutant cell lines and patient-derived myeloid blasts (MDS/AML), showing a heightened stress granule response.
Conclusions:
- U2AF1 mutations directly alter 3'SS recognition, leading to widespread splicing dysregulation.
- The study reveals a novel link between U2AF1 mutations, splicing defects, and the formation of stress granules.
- Biomolecular condensates, such as stress granules, may play an adaptive role in oncogenesis driven by splicing factor mutations.
