Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Time-resolved profiling of mRNA-protein interaction through mRNA life cycle

ABSTRACT: From synthesis to decay, mRNA changes its protein partners, yet previous studies were limited in that they profiled only the mixed pools of RNA-binding proteins (RBPs) without temporal resolution. Here, we provide time-resolved profiles of human mRNA interactome by combining pulse metabolic labeling, photoactivatable ribonucleoside crosslinking, poly(A) RNA isolation, and mass spectrometry. We captured stage-specific RBPs across 10 time points and quantified over 700 RBPs. The chronological orders of mRNA binding are generally consistent with the known functions and localizations of RBPs: pre-mRNA processing factors are detected as “early binders” while decay factors appear as “late binders”. Notably, stress granule proteins are enriched in “aged” mRNPs, suggesting their roles in the final stage of mRNA life cycle. Many late binders also interact with viral transcripts, implying their regulatory activities on viruses. Furthermore, we built a computational model to systematically identify RBPs with unexpected binding dynamics, which indicates their unknown functions. Our study reveals a dynamic landscape of mRNP remodeling, offering new functional insights into RNA-protein interaction during mRNA life cycle.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Yeon Choi

LAB HEAD: V. Narry Kim

PROVIDER: PXD039054 | Pride | 2024-02-23

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Deuterium-free, Three-plexed Peptide Di-Ethylation for Highly Accurate Quantitative Proteomics

Project description:The deuterium, frequently used stable isotope in isotopic labeling for quantitative proteomics, could deteriorate the accuracy and precision of proteome quantification owing to the retention time shift of deuterated peptides from the hydrogenated counterpart. Herein, we introduce a novel three-plexed peptide ‘di-ethylation’ using only 13C-isotopologues of acetaldehyde and demonstrate that the accuracy and precision of our method in proteome quantification are significantly superior to the conventional deuterium-based di-methylation labeling in both a single shot and multidimensional LC-MS/MS analysis of HeLa proteome. Furthermore, in time-resolved profiling of Xenopus laevis early embryogenesis, our 3-plexed di-ethylation outperformed isobaric labeling approaches in terms of quantification accuracy or number of protein identification, generating >2 times more differentially expressed proteins. Our cost-effective and highly accurate 3-plexed di-ethylation method could contribute to various types of quantitative proteomics applications, in which three of multiplexity would be sufficient.

2019-01-17 | PXD012117 | Pride

β-actin RNA-BioID

Project description:Function and fate of mRNAs is controlled by RNA binding proteins (RBPs) but determining the proteome of a specific mRNA in vivo is still challenging. RNA proximity biotinylation on the transported β-actin mRNA tagged with MS2 aptamers (RNA-BioID) is used to characterize the dynamic proteome of the β-actin mRNP in mouse embryonic fibroblasts (MEFs). We have identified > 60 β-actin associated RBPs including all six previously known as well as novel interactors. By investigating the dynamics of the β-actin mRNP in MEFs, we expand the set of β-actin mRNA associated RBPs and characterize the changes of the interacting proteome upon serum-induced mRNA localization. We report that the KH-domain containing protein FUBP3 represents a new β-actin associated RBP that binds to its 3’-untranslated region outside the known RNA localization element but is required for β-actin RNA localization. RNA-BioID will allow to obtain a dynamic view on the composition of endogenous mRNPs.

2020-03-12 | PXD010694 | Pride

Transcriptome-wide identification of coding and noncoding RNA-binding proteins defines the extended RNA interactome of Leishmania mexicana

Project description:Non-poly(A) RNA molecules including noncoding RNAs (ncRNAs) comprise the major portion of the total transcribed molecules in the cell. In addition to the mRNAs the ncRNAs also function as ribonucleoprotein particles (RNPs) and carry out biological functions including synthesis of new proteins, RNA processing, genome remodelling and regulation of transcription. We therefore envisaged a comprehensive transcriptome-wide identification of coding and non-coding RNA-binding proteins (RBPs) in the Leishmania spp. Towards this we applied the recently reported orthogonal organic phase separation (OOPS) method in combination with tandem mass tag (TMT) labelling-based quantitative proteomic mass spectrometry (MS) and report herein the most comprehensive identification of RBPs in Leishmania mexicana (L. mexicana) parasites. This study identified novel RNA binding property of thousands of L. mexicana proteins, significantly expanding the RBP landscape of the parasite. Furthermore, we showed that the classical Hsp90 inhibitor tanespimycin differentially regulates the RNA-binding property of hundreds of L. mexicana RBPs, shedding light into hitherto unknown large-scale downstream molecular effects of the small molecule inhibitor in the parasite.

2022-01-14 | PXD028092 | Pride

Quantitative Proteomics to identify nuclear RNA-binding proteins of Malat1

Project description:The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function in the context of RBPs identification of their RNA-binding proteins can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5’-region and one in the 3’-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.

2020-01-29 | PXD017309 | Pride

Profile of the SARS-CoV-2 RNA interactome

Project description:SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

2021-04-28 | PXD024808 | Pride

Identification of cellular proteins binding to the 3’UTR of Programmed Cell Death 4 mRNA

Project description:The aim of the project is to identify RNA-binding proteins (RBPs) that interact with the 3’ untranslated region (3’UTR) of the programmed cell death 4 (PDCD4) mRNA. PDCD4 is an inflammatory tumor suppressor gene. The translation of PDCD4 mRNA is regulated by multiple RBPs. The aim is to identify RBPs which are critical for regulating PDCD4 mRNA translation by binding to its 3’UTR. For this purpose, a biotinylated PDCD4 3’UTR RNA was incubated with cytoplasmic lysate from MCF7 human breast carcinoma cells and the RNA-protein complexes pulled down by streptavidin affinity chromatography. The proteins were then eluted and resolved by SDS-PAGE. One particular protein band, migrating close to 55 KDa marker was cut out and submitted for proteomic analysis by in gel digestion and mass spectrometry.

2020-12-11 | PXD017376 | Pride

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro (XL-MS)

Project description:SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic, entailing enormous disruption worldwide. Its ~30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein, and the data suggest that the nsp7-10/11 structure in complex with Mpro strongly resembles the unbound polyprotein. Our array of techniques supports the notion that interplay between polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we leveraged assays of limited proteolysis by Mpro of nsp7-11 using a series of inhibitors/binders to characterize Mpro inhibition using a polyprotein substrate.

2022-12-19 | PXD033748 | Pride

rec-Y2H matrix in S. cerevisiae screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Project description:RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs recognized by RBPs are typically a poor predictor of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory potential. To probe the potential for RBP–RBP complex formation, we assembled a library of 978 mammalian RBPs and used rec-Y2H screening to detect direct interactions between RBPs, sampling >1M possible interactions. We discovered 1994 new interactions and demonstrate that our interaction screening discovers RBP pairs that bind RNAs adjacently. We further find that the mRNA binding region preferences of an RBP can deviate, depending on its adjacently binding interaction partner. Finally, we reveal novel RBP–RBP interaction networks among major RNA processing steps and show that RBP mutations observed in cancer rewire spliceosomal interaction networks.

2021-09-01 | E-MTAB-9612 | biostudies-arrayexpress

The RNA-binding protein RBP42 regulates cellular energy metabolism in mammalian-infective Trypanosoma brucei

Project description:RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long unrelated gene arrays. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective bloodstream form of African trypanosome, and show that RBP42 is a key regulator of parasite’s central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Global quantitative transcriptomic and proteomic analyses reveal that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a positive translational regulatory role of RBP42. Significant changes in central carbon metabolic intermediates, following loss of RBP42, further support its critical role in cellular energy metabolism.

2021-06-11 | PXD026622 | Pride

Chronically elevated exogenous glucose elicits antipodal effects on the proteome signature of differentiating human pancreatic progenitors

Project description:The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is thus extremely valuable for managing the disease progression. Extracellular glucose is known to impact cell identity by impacting the redox balance. Here we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, high exogenous glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex yin-yang action of exogenous glucose on differentiating pancreas progenitors with a distinct proteome signature.

2021-09-09 | PXD022177 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data