Biology subjects

Choder, M.

Publications and source records attributed to Choder, M..

6 recordsLinked to original sources

bioRxiv2026

mRNA Imprinting: transcription apparatus can remotely control cytoplasmic post-transcriptional mechanisms by dozens of proteins

Proper regulation of gene expression requires the harmonious coordination of transcription with other stages of the mRNA lifecycle. We have previously demonstrated that proteins can bind to nascent transcripts co-transcriptionally - a phenomenon named "mRNA imprinting" because the bound proteins modulate subsequent stages of the mRNA lifecycle. Using a novel high-throughput approach, "PRofiling OF Imprinted Transcripts" ("PROFIT"), to identify proteins involved in mRNA imprinting, we uncovered several dozen candidates. The RNA polymerase II (Pol II) subunit Rpb4, which can itself imprint mRNAs, mediates the imprinting of a large subset of these proteins. Imprinting repertoire and profile is responsive to environmental changes and include HSP70 variants. Interestingly, PROFIT identified proteins previously thought to function mainly or exclusively in the cytoplasm, including a translation factors, mRNA decay factor, protein chaperones, substrate-delivering factors of the proteasome, targeting factors that deliver mRNAs to mitochondria and more. Using proximity labeling, we validated several hits, including two translation initiation factors: eIF4G and the eIF3 component Rpg1. Importantly, we found that the same Rpg1 molecule, which had been transiently localized near Pol II, co-sediments with polyribosomes - similar to the bulk Rpg1. Our results suggest that the transcription machinery can regulate translation by recruiting specific translation factors, which later participate in protein synthesis. mRNA imprinting appears to be a widespread phenomenon, and we speculate that it may not be limited to the transcription stage alone. Interestingly, the PROFIT experiments identified proteins with known cytoplasmic function, including a translation factor (Rpg1), mRNA decay factor (Xrn1), protein chaperones (Ssa1/2), substrate-delivering factors of the proteasome, targeting factors that deliver mRNAs to mitochondria, and actin-binding factors.

molecular biology↗

bioRxiv2023

Fine-tuning Rpb4 phosphorylation modulates Rpb4/7 stoichiometry within yeast RNA polymerase II and regulates gene expression

RNA polymerase II (RNAPII) consists of 12 subunits, including Rpb4 and Rpb7, which form a heterodimeric stalk domain. In yeast, assembly of Rpb4/7 with the 10-subunit "core" is substoichiometric. Aside from its role in transcription, Rpb4/7 also binds to mRNAs co-transcriptionally (mRNA imprinting), exits the nucleus together with the mRNA, and modulates subsequent cytoplasmic processes like translation and degradation. However, the mechanisms underlying Rpb4/7 functions and its association with RNAPII remain poorly understood. In this study, we identify five phosphorylation sites in Rpb4 that regulate various steps of mRNA and snoRNA biogenesis, including transcription elongation and the polyadenylation step. We also provide evidence that the kinase Hrr25 and the phosphatase Fcp1, known to target the carboxyl-terminal domain (CTD) of Rpb1 and participate in processes at gene 3'-ends, coordinate Rpb4 phosphorylation. Inefficient Rpb4 phosphorylation leads to increased Rpb4 association with RNAPII, particularly at gene 3'-ends, suggesting that Rpb4-P promotes stalk dissociation from RNAPII either before or during transcription termination. Collectively, our findings underscore the pivotal role of dynamic Rpb4 phosphorylation in regulating Rpb4/7 stoichiometry as well as transcription elongation and termination, with an impact on mRNA imprinting.

molecular biology↗

bioRxiv2023

The transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis and cytoplasmic decay

To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here we focus on Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1s dissociation from chromatin. . Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1s relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1s co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs. The interplay between Sfp1s two modes of transcription regulation remains to be examined.

molecular biology↗

bioRxiv2021

RNA-controlled nucleocytoplasmic shuttling of mRNA decay factors regulates mRNA synthesis and initiates a novel mRNA decay pathway

mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the exonuclease Xrn1 - a major mRNA synthesis and decay factor. Here we show that nucleocytoplasmic shuttling of Xrn1 and of some of its associated mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA-controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Mutations in the two NLSs, which prevent Xrn1 import, compromise transcription and, unexpectedly, also the cytoplasmic decay of [~]50% of the cellular mRNAs - comparably to Xrn1 deletion. These findings uncover a cytoplasmic mRNA decay pathway that begins in the nucleus. Interestingly, Xrn1 shuttling is required for proper adaptation to environmental changes, in particular to ever changing environmental fluctuations.

molecular biology↗

bioRxiv2019

Numerous post-translational modifications of RNA polymerase II subunit Rpb4 link transcription to post-transcriptional mechanisms

Rpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., Pol II, eIF3, Pat1) both temporarily and spatially, Rpb4/7 regulates the major stages of the mRNA lifecycle. Here we show that Rpb4/7 can undergo over 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTMs repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. A mutagenesis approach in residues that undergo PTMs suggests that temporal Rpb4 PTMs regulate its interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis despite its normal association with Pol II, whereas the other affects both mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay ( mRNA buffering) and the cells capacity to respond to the environment. Taken together, we propose that temporal Rpb4/7 PTMs are involved in cross talks among the various stages of the mRNA lifecycle.

molecular biology↗

bioRxiv2019

Cytoplasmic mRNA decay factors modulate RNA polymerase II processivity in 5’ and 3’ gene regions in yeast

mRNA levels are determined by the balance between mRNA synthesis and decay. Factors that mediate both processes, including the 5 to 3 exonuclease Xrn1, are responsible for the cross talk between the two processes in a manner that buffers steady-state mRNA levels. However, these proteins roles in transcription remain elusive and controversial. Applying NET-seq to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests via the reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. We combine our data and novel mathematical modeling of transcription to suggest that transcription initiation and elongation of targeted genes is modulated by Xrn1. Furthermore, Pol II occupancy markedly increases near cleavage and polyadenylation sites in xrn1{Delta} cells while its activity decreases, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. Two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, while the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, DFs are capable of differentiating between SAGA- and TFIID-dominated promoters. These two classes of genes respond differently to XRN1 deletion in mRNA synthesis and differentially utilize mRNA decay pathways, raising the possibility that one distinction between the two types of genes lies in the mechanism(s) that balance these processes.

genomics↗