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CacyBP/SIP - RPL6 interaction: potential influence on ribosome function

ABSTRACT: Previously, we have shown that CacyBP/SIP interacts with NPM1, a protein involved in ribosome biogenesis. In this work, examined the potential impact of CacyBP/SIP on ribosome biogenesis and/or function. At first, we searched for ribosomal proteins (RPs) that might interact with CacyBP/SIP. Using mass spectrometry analysis, we have found that several RPs could be potential CacyBP/SIP targets. Then, by applying various biochemical methods, we confirmed that the interaction between CacyBP/SIP and RPL6 was direct. Moreover, in silico analysis allowed us to establish the domains/fragments of both proteins involved in this interaction. Subsequently, we performed several analyses using neuroblastoma NB2a cell line with stably silenced CacyBP/SIP expression. We have found, by applying OPP (O-propargyl-puromycin), that the number of cells with protein synthesis signal ("translation signal") was significantly lower in the cell line with diminished CacyBP/SIP level than in control one. Moreover, we observed lower induction of the synthesis of Hsp70, a stress-inducible protein, in NB2a cells with diminished CacyBP/SIP level after heat shock. In parallel, we have shown that CacyBP/SIP may have an impact on ER architecture/network. This confirms the influence of CacyBP/SIP, most probably through interaction with RPL6 and/or other RPs, on ribosome function and, in consequence, on protein synthesis in the cell.

INSTRUMENT(S): LTQ Orbitrap Velos, Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Agata Malinowska

LAB HEAD: Ewelina Jurewicz

PROVIDER: PXD064560 | Pride | 2025-07-28

REPOSITORIES: Pride

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Project description:DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding and high-throughput sequencing (PB-seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high throughput sequencing. We compared PB-seq binding profiles with ones observed in vivo by ChIP-seq, and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity. We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome. HSF-bound DNA was specifically eluted and detected by high throughput sequencing. Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site. The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope. Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications: HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column. We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature. We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA. The mock IP was done in the absence of recombinant HSF.

			Action	DRS
	208063234maks_IP_FLAG.raw	Raw
	208063235maks_IP_Cacy.raw	Raw
	512172895rosi_Flag.raw	Raw
	512172897rosi_SIP.raw	Raw
	60127111rosi_FLAG.raw	Raw

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CacyBP/SIP - RPL6 interaction: potential influence on ribosome function

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