Clements, J. L. | Search bioRxiv

bioRxiv2026

HDAC11 Regulates RNA Splicing via De-Fatty Acylation of SF3B2

Histone deacetylase 11 (HDAC11) is a lysine de-fatty acylase whose cellular substrates and mechanisms remain incompletely defined. Here, using metabolic labeling, mass spectrometry, click chemistry, and standard molecular biology, we show that SF3B2 is modified by lysine myristoylation at K10 and that HDAC11 efficiently removes this modification in cells, establishing SF3B2 as a direct enzymatic substrate. A de-myristoylation mimetic mutant (SF3B2 K10R) exhibits altered pre-mRNA binding activity in a context-dependent manner. In HCC cells, loss of SF3B2 lysine myristoylation enhances SF3B2 association with androgen receptor (AR) splice variant loci and promotes alternative splicing towards the AR-v7 variant. Consistently, HDAC11 overexpression increases, and HDAC11 knockdown decreases, the AR-v7/AR-FL splice isoform ratio in HCC cells in a manner requiring HDAC11 catalytic activity and recapitulated by SF3B2 K10R. In contrast, modulation of HDAC11 does not alter AR splicing in prostate cancer cells, indicating cell type specific regulation. Together, these findings establish lysine myristoylation as a reversible regulatory modification on a spliceosomal component and reveal HDAC11-catalyzed de-myristoylation of SF3B2 as a mechanism that can tune alternative splicing in liver cancer cells. In BriefClements et al. utilize metabolic labelling, mass spectrometry, click chemistry, and protein and RNA biochemistry to establish that a histone deacetylase enzyme, HDAC11, can influence RNA splicing through de-fatty acylation of the RNA splicing factor SF3B2. De-fatty acylation of SF3B2 at K10 by HDAC11 modulates SF3B2s pre-mRNA binding to AR splice variant loci, thereby driving alternative splicing of the AR-v7 variant in a cell type dependent manner. This work provides direct mechanistic evidence linking an HDAC to RNA splicing, identifies a reversible lipid modification on SF3B2, and expands current understanding of post-translational regulation of spliceosomal proteins and HDAC11 de-fatty acylation substrates. HighlightsO_LIHDAC11 de-fatty-acylates SF3B2 at K10, revealing a previously unrecognized modification on SF3B2. C_LIO_LISF3B2 de-fatty acylation enhances alternative splice-site binding in liver cancer cells. C_LIO_LIHDAC11 regulates RNA splicing through enzymatic de-fatty acylation of a spliceosomal protein. C_LI

molecular biology↗

bioRxiv2019

Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation

Stimulation of receptor tyrosine kinases (RTK) such as EGF locally increase reactive oxygen species (ROS) levels at the plasma membrane that oxidize cysteines in proteins to enhance downstream signaling. Spatial confinement of ROS is an important regulatory mechanism to redox signaling, but it remains unknown why stimulation of different receptor tyrosine kinases (RTKs) at the plasma membrane target distinct sets of downstream proteins. To uncover additional mechanisms specifying which cysteines are redox regulated by EGF stimulation, we performed time-resolved quantification of the oxidation of 4,200 cysteine sites subsequent to EGF stimulation in A431 cells. EGF induces three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamic simulation indicate widespread redox regulation of cryptic cysteines that are only solvent exposed upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates serve as two distinct modes by which EGF specifies which cryptic cysteines become solvent exposed and redox regulated. Since proteins structurally regulated by different RTKs or cellular perturbations are largely unique, solvent exposure and redox regulation of cryptic cysteines is an important mechanism contextually delineating redox signaling networks.\n\nSignificance StatementCellular redox processes are interconnected, but are not in equilibrium. Thus, understanding the redox biology of cells requires a systems-level, rather than reductionist, approach. Factors specifying which cysteines are redox regulated by a stimulus remain poorly characterized but are critical to understanding the fundamental properties of redox signaling networks. Here, we show that EGF stimulation induces oxidation of specific cysteines in 3 distinct spatiotemporal patterns. Redox regulated proteins include many proteins in the EGF pathway as well as many cysteines with known functional importance. Many redox regulated cysteines are cryptic and solvent exposed by changes in protein structure that were induced by EGF treatment. The novel finding that cryptic cysteines are redox regulated has important implications for how redox signaling networks are specified and regulated to minimize crosstalk. In addition, this time-resolved dataset of the redox kinetics of 4,200 cysteine sites is an important resource for others and is an important technological achievement towards systems-level understanding of cellular redox biology.

biochemistry↗