Publications by authors named "Mike Fernandez"
Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis.
Cancer Res Commun
September 2024
Article Synopsis
- SF3B1 is a critical RNA splicing gene frequently mutated in cancer, but studying its pathogenic effects has been challenging due to the lack of suitable cell line models.
- By comparing various genome editing techniques, the study found that prime editing, specifically using PE5max, was the most effective method for introducing the SF3B1 K700E mutation in multiple cell lines.
- The researchers introduced this mutation into chronic lymphocytic leukemia cell lines using a novel approach called PRECIS, which not only mimicked key mutant characteristics but also revealed new insights into the mutation's role in cancer progression and genome instability.
RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear.
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- RNA splicing dysregulation plays a crucial role in the development and progression of chronic lymphocytic leukemia (CLL), affecting about 20% of patients with spliceosome mutations.
- An analysis revealed that proteins involved in RNA splicing are increasingly produced in CLL cells, leading to splicing dysregulation which is linked to a poor prognosis.
- The study highlights METTL3, an RNA methyltransferase, as a key factor influencing splicing factor expression through m6A modifications and suggests it could be targeted for new treatments in aggressive CLL cases.
The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA.
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- The study focuses on how specific protein factors recognize splice sites in mRNA, particularly highly degenerate ones, by binding to single-stranded exonic splicing motifs while lacking detailed understanding of their secondary structures.
- Analysis of mouse transcriptomes reveals that retained introns are flanked by a base-paired segment upstream and a single-stranded exonic segment downstream, with upstream loops containing splicing enhancers that are crucial for proper splicing.
- Substituting or hybridizing these upstream loops hinders splicing, indicating that the structure of exonic segments can regulate alternative splicing by affecting the accessibility of necessary splicing factors.