Biology subjects

Shchepachev, V.

Publications and source records attributed to Shchepachev, V..

6 recordsLinked to original sources

bioRxiv2024

Pin4 Links Post-transcriptional and Transcriptional Responses to Glucose Starvation in Yeast

Adaptation to environmental change is essential in all organisms, with RNA-binding proteins (RBPs) playing critical roles in rapid cellular responses. We analyzed the largely uncharacterized yeast RBP Pin4, and its involvement in adaptation to glucose depletion. UV crosslinking (reCRAC) revealed that in glucose conditions Pin4 binds mRNA 3 UTRs with preference for a specific motif in mRNAs involved in glycolysis, amino acid, and mitochondrial metabolism. Following glucose withdrawal, Pin4-RNA binding was greatly reduced, with residual binding favoring transcripts associated with protein translation. Cells lacking Pin4 were greatly impaired in recovery from nutrient starvation and hypersensitive to oxidative stress, consistent with the mRNA targets. RNAseq and reporter assays indicated that loss of Pin4 correlated with some increases in target mRNA abundance. In wildtype yeast, glucose depletion induces diauxic shift, with massive changes in transcription patterns. Unexpectedly, this response was almost entirely abolished in cells lacking Pin4, its C-terminal prion-like domain, the RNA-recognition motif (RRM) or with an RRM point mutation. Pin4 is implicated in sensing energy depletion, which also occurs during the approach to stationary phase. We postulate that Pin4 helps coordinate post-transcriptional and transcriptional responses to energy stress, via riboregulation.

cell biology↗

bioRxiv2024

reCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and non-specific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.

molecular biology↗

bioRxiv2021

A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described. HighlightsO_LIMutations in RMRP lncRNA impair pre-rRNA processing and T cell activation C_LIO_LIPatient derived fibroblasts show impaired pre-rRNA processing C_LIO_LICells with the most common disease-linked mutation have specific processing defects C_LIO_LICytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells C_LI

cell biology↗

bioRxiv2020

Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

cell biology↗

bioRxiv2020

Stress-induced translation inhibition through rapid displacement of scanning initiation factors

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA-association. Among translation components, RNA-association was most reduced for initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5'-ends of mRNAs. Following glucose withdrawal, 5-binding was abolished within 30sec, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5 RNA-binding by initiation factors over [~]16min. Translation shutoff provoked selective 5'-degradation of mRNAs encoding translation-related factors, mediated by Xrn1. These results reveal mechanisms underlying translational control of gene expression during stress. HighlightsA quantitative proteomic approach reveals rapid stress-induced changes in RNA-binding Translation shutdown is driven by loss of mRNA binding by scanning initiation factors eIF4B and Ded1 have key but separate roles in driving the stress response Heat shock invokes rapid RNA degradation by Xrn1, selective for translation machinery

cell biology↗

bioRxiv2018

Defining the RNA Interactome by Total RNA-Associated Protein Purification

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

cell biology↗