Biology subjects

Vayyeti, A.

Publications and source records attributed to Vayyeti, A..

3 recordsLinked to original sources

bioRxiv2026

A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs

The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.

biochemistry↗

bioRxiv2025

Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein that is integral to DNA metabolism. Segregation of RPA functions in response to DNA damage is fine-tuned by hyperphosphorylation of the RPA32 subunit that is dependent on Cyclin-dependent kinase (Cdk)-mediated priming phosphorylation at the Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation of RPA remains unresolved. Furthermore, the modulation of cell cycle progression by the RPA-Cdk axis is not clearly understood. Here, we uncover that the RPA70 subunit is also phosphorylated by Cdk1 at Thr-191. This modification is crucial for the G2 to M phase transition. This function is enacted through reciprocal regulation of Cdk1 activity through a feedback circuit espoused by stabilization of Wee1 kinase. The Thr-191 phosphosite on RPA70 is also crucial for priming hyperphosphorylation of RPA32 in response to DNA damage. Structurally, phosphorylation by Cdk1 primes RPA by reconfiguring the domains to release the N-terminus of RPA32 and the two protein-interaction domains that markedly enhances the efficiency of multisite phosphorylation by other kinases. Our findings establish a unique phosphocode-dependent feedback mechanism between RPA and RPA-regulating kinases that is fine-tuned to enact bipartite functions in cell cycle progression and DNA damage response.

biochemistry↗

bioRxiv2023

Fluorescent human RPA to track assembly dynamics on DNA

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Forster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. HighlightsO_LIRPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination. C_LIO_LIRPA is a ssDNA binding protein made of six domains situated across RPA70, RPA32 and RPA14 subunits. Four high affinity DNA binding domains engage the DNA. C_LIO_LISite-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. C_LIO_LIBulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA. C_LI

biochemistry↗