Biology subjects

Sanford, J. R.

Publications and source records attributed to Sanford, J. R..

5 recordsLinked to original sources

bioRxiv2024

Metabolic regulation of RNA methylation by the m6A-reader IGF2BP3

The interplay of RNA modifications - deposited by "writers", removed by "erasers" and identified by RNA binding proteins known as "readers" - forms the basis of the epitranscriptomic gene regulation hypothesis. Recent studies have identified the oncofetal RNA-binding protein IGF2BP3 as a "reader" of the N6-methyladenosine (m6A) modification and crucial for regulating gene expression. Yet, how its function as a reader overlaps with its critical oncogenic function in leukemia remains an open question. Here, we report the novel finding that the reader IGF2BP3 reprograms cellular metabolism, resulting in an altered ability of the "writers" to modify the epitranscriptome. In leukemia cells, IGF2BP3 supports increased glycolytic flux and one-carbon metabolism, leading to increased production of S-adenosyl methionine (SAM), a key substrate for methylation reactions within the cell. IGF2BP3 directly regulates the translation of MAT2B, the regulatory subunit of the methionine-adenosyltransferase complex, which is the final enzyme in a pathway leading to SAM production. This, in turn, results in increased m6A modifications on RNA, resulting in positive feedback regulation. This novel mechanism illustrates how metabolism mutually acts with epitranscriptomic modifications, underscoring the pervasive impact of IGF2BP3 in gene regulatory mechanisms governing a broad range of cancer-specific processes.

cancer biology↗

bioRxiv2023

Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides

The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 ([~]14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 ([~]36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3' end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3' splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3 splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.

molecular biology↗

bioRxiv2020

Post-transcriptional gene regulation by the RNA binding protein IGF2BP3 is critical for MLL-AF4 mediated leukemogenesis

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes and a high risk of relapse. Here, we found that MLL-AF4, the most common MLL fusion protein in patients, transcriptionally induces IGF2BP3 and that IGF2BP3 strongly amplifies MLL-Af4 mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4 driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. eCLIP and transcriptome analysis of MLL-Af4 transformed stem and progenitor cells and MLL-Af4 bulk leukemia cells reveals a complex IGF2BP3-regulated post-transcriptional operon governing leukemia cell survival and proliferation. Regulated mRNA targets include important leukemogenic genes such as those in the Hoxa locus and numerous genes within the Ras signaling pathway. Together, our findings show that IGF2BP3 is an essential positive regulator of MLL-AF4 mediated leukemogenesis and represents an attractive therapeutic target in this disease.

cancer biology↗

bioRxiv2020

Splicing factor SRSF1 expands the regulatory logic of microRNA expression

The serine and arginine-rich splicing factor SRSF1 is an evolutionarily conserved, essential pre-mRNA splicing factor. Through a global protein-RNA interaction survey we discovered SRSF1 binding sites 25-50nt upstream from hundreds of pre-miRNAs. Using primary miRNA-10b as a model we demonstrate that SRSF1 directly regulates microRNA biogenesis both in vitro and in vivo. Selective 2 hydroxyl acylation analyzed by primer extension (SHAPE) defined a structured RNA element located upstream of the precursor miRNA-10b stem loop. Our data support a model where SRSF1 promotes initial steps of microRNA biogenesis by relieving the repressive effects of cis-regulatory elements within the leader sequence.

molecular biology↗

bioRxiv2017

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3 splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3 splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to \"decoy\" binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3 splice sites.

molecular biology↗