Biology subjects

Boutz, P. L.

Publications and source records attributed to Boutz, P. L..

7 recordsLinked to original sources

bioRxiv2026

CDK12 and CDK13 suppress distinct intronic polyadenylation sites

Cyclin-dependent kinases CDK12 and CDK13 are RNA polymerase II C-terminal domain Ser2 kinases that regulate transcriptional elongation and co-transcriptional RNA processing. Loss-of-function (LOF) mutations in these kinases are observed in multiple cancers and are characterized by increased intronic polyadenylation (IPA), which generates prematurely terminated transcripts and can give rise to novel intronic peptides. To investigate the distinct contributions of CDK12 and CDK13 to suppressing early transcriptional termination, we modeled selective kinase inhibition in OVCAR4 ovarian cancer cells using THZ531, a dual CDK12/13 inhibitor. A CRISPR-engineered CDK12 C1039S mutation conferred THZ531 resistance, enabling functional distinction between CDK12 and CDK13 LOF conditions. Using Poly(A) Click-seq (PACseq) across a six-point inhibitor dose curve, we identified thousands of IPA sites with distinct dose-response behaviors, allowing us to classify CDK12- and CDK13-dependent subsets. Oxford Nanopore long-read sequencing confirmed premature termination events and revealed full-length isoform switches, demonstrating how IPA alters coding potential by truncating protein domains and producing novel, intronically encoded peptide sequences. In parallel, integration of long-read transcriptomes with mass spectrometry data identified peptides derived from IPA isoforms, confirming that intronic sequences can be translated. Together, these findings establish distinct roles for CDK12 and CDK13 in maintaining transcript integrity, demonstrate that IPA transcripts are not only abundant but also translationally competent, and provide evidence for a mechanistic link between transcriptional dysregulation and the generation of novel peptide isoforms in CDK12/13-deficient cancers. These results highlight a potential therapeutic vulnerability through the exploitation of IPA-derived peptides as tumor-specific neoantigens.

genomics↗

bioRxiv2026

Phosphorylation-mediated regulation of the essential splicing factor PUF60

PUF60 is a splicing factor related to the polypyrimidine-tract binding protein U2AF2. PUF60 is deleted in developmental disorders such as Verheij syndrome and amplified in approximately 8% of cancers. Thus, both increases and decreases in PUF60 expression can have profound physiological effects. However, little is known about how changes in PUF60 expression impact global splicing patterns. Here, we created a model system of CRISPRa/i in mouse stem cells (mESCs) to transcriptionally upregulate or downregulate Puf60. Our results uncovered extensive transcriptional, post-transcriptional, and post-translational regulation of Puf60 protein expression. We observed that Puf60 protein levels in normal mESCs drop dramatically at a critical cell density, leading to cell death. Puf60 is very essential in stem cells, and its repression causes cell death and impacts specific splicing events, including its own splicing autoregulation, providing valuable insights into the functional consequences of PUF60 dysregulation. Analysis of phosphoprotein data revealed phosphorylation of threonine at the N-terminus of PUF60. Our results showed that mutating threonine to glutamate downregulates the protein and alters its localization. Thus, our study reveals a novel regulatory mechanism of Puf60 phosphorylation that mediates its function and may be related to its frequent overexpression in cancer cells.

molecular biology↗

bioRxiv2026

SF3B1 Phosphorylation Prompts U2AF2 Dissociation for Widespread Control of pre-mRNA Splicing

Reversible pre-mRNA splicing factor phosphorylation is a well-documented feature of spliceosome assembly, activation, and disassembly, yet its functional and mechanistic roles are still emerging. SF3B1, a core spliceosome subunit, is extensively phosphorylated before the first catalytic step of pre-mRNA splicing. Intriguingly, most SF3B1 phosphorylation sites surround its binding sites for U2AF2, an early-stage pre-mRNA splicing factor. Here, we discovered that SF3B1 phosphorylation significantly decreases its association with U2AF2. We determined two crystal structures revealing electrostatic repulsion between an acidic U2AF2 -helix and the negatively-charged phosphoryl group of SF3B1. Variants with amino acid substitutions that prevent or mimic SF3B1 phosphorylation perturbed thousands of splice sites, primarily those marked by uridine-rich splice site signals recognized by U2AF2. Collectively, our findings demonstrate a widespread but previously unrecognized role for SF3B1 phosphorylation as a gateway for U2AF2 dissociation so that pre-mRNA splicing can proceed.

biochemistry↗

bioRxiv2025

Recurrent oncogenic ZC3H18 mutations stabilize endogenous retroviral RNA

Endogenous retroviral (ERV) RNA is highly expressed in cancer, although the molecular causes and consequences remain unknown. We found that ZC3H18 (Z18), a component of multiple nuclear RNA surveillance complexes, has recurrent truncating mutations in cancer. We show that Z18trunc mutations are oncogenic and that Z18 plays an evolutionarily conserved role in nuclear RNA surveillance of ERV RNA. In zebrafish, Z18trunc expedited melanoma onset and promoted a specific accumulation of ERV RNA. Z18 mutant human cell lines from the Cancer Cell Line Encyclopedia also expressed higher levels of ERV RNA. In engineered human melanoma cells, Z18trunc enhanced ERV RNA accumulation more than loss of one Z18 copy, indicating dominant negative activity. Z18trunc directly bound and stabilized ERV RNA. Notably, expression of ERV RNA was sufficient to expedite oncogenesis in a zebrafish model, which is the first evidence of which we are aware that ERV transcripts can play a functional role in cancer. Our work illuminates a mechanism for elevated ERV transcripts in cancer and supports that aberrant RNA accumulation is broadly oncogenic.

cancer biology↗

bioRxiv2024

The PRMT5-splicing axis is a critical oncogenic vulnerability that regulates detained intron splicing

Protein arginine methyltransferase 5 (PRMT5) is a promising cancer target, yet its unclear which PRMT5 roles underlie this vulnerability. Here, we establish that PRMT5 inhibition induces a special class of unspliced introns, called detained introns (DIs). To interrogate the impact of DIs, we depleted CLNS1A, a PRMT5 cofactor that specifically enables Sm protein methylation. We found that many, but not all, cell lines are CLNS1A-dependent and established that loss of viability is linked to loss of Sm protein methylation and DI upregulation. Finally, we discovered that PRMT5-regulated DIs, and the impacted genes, are highly conserved across human, and also mouse, cell lines but display little interspecies conservation. Despite this, human and mouse DIs have convergent impacts on proliferation by affecting essential components of proliferation-regulating complexes. Together, these data argue that the PRMT5-splicing axis, including appropriate DI splicing, underlies cancers vulnerability to PRMT5 inhibitors.

cancer biology↗

bioRxiv2022

A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

In the early stages of spliceosome assembly, the 3 splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 [A] resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the fulllength proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

biochemistry↗

bioRxiv2019

CDK13 Mutations Drive Melanoma via Accumulation of Prematurely Terminated Transcripts

Transcriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

biophysics↗