Dataset Information

MRNA length and 3’UTR structure govern the translation selectivity in RPS14-haploinsufficient erythroblasts

ABSTRACT: 5q- syndrome is a somatic ribosomopathy linked to the monoallelic deletion of the RPS14 gene and characterized by a proeminent erythroid phenotype. The mechanism of anemia involves an impaired differentiation and increased apoptosis of erythroblasts. We have analyzed total cell extracts from UT7 cells with or without a decrease of RPS14 proteins induced by shRNAs. Our data show that GATA1 protein expression is low in line with a defect in the representation of its mRNA at the ribosome. A global analysis of transcripts on polysomes indicates that translation is selective with a decreased representation of the transcripts with a short coding sequence and UTRs and a highly structured 3’UTR, a subset of transcripts that includes GATA1. Our whole proteome analysis confirms that post-transcriptionally downregulated proteins were encoded by transcripts with a short length and structured 3’UTR. We identified a subset of post-translationally downregulated proteins including ribosomal proteins and translation elongation factors encoded by 5’TOP mRNAs that were enriched on the ribosome. Our results indicate that the thermodynamic characteristics of 3’UTR and in a lesser extend 5’UTR and the transcript length are the determinants of translation selectivity under RPS14 haploinsufficiency conditions and that a post-translational regulation of ribosomal proteins accounts for their decreased content in the cell.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Blood Cell, Permanent Cell Line Cell, Blood

DISEASE(S): Acute Leukemia

SUBMITTER: Emilie-Fleur GAUTIER

LAB HEAD: Patrick MAYEUX

PROVIDER: PXD008650 | Pride | 2021-04-28

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	MiFO170522_UT7-640-avec-Dox_F21283.raw	Raw
	MiFO170522_UT7-641-avec-Dox_F21392.raw	Raw
	MiFO170522_UT7-SCR-avec-Dox_F21281.raw	Raw
	MiFO170704_640Doxplus_SCX1_F22467.raw	Raw
	MiFO170704_640Doxplus_SCX2_F22468.raw	Raw

Items per page:

1 - 5 of 23

Publications

Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells.

Boussaid Ismael I Le Goff Salomé S Floquet Célia C Gautier Emilie-Fleur EF Raimbault Anna A Viailly Pierre-Julien PJ Al Dulaimi Dina D Burroni Barbara B Dusanter-Fourt Isabelle I Hatin Isabelle I Mayeux Patrick P Cosson Bertrand B Fontenay Michaela M

Haematologica 20210301 3

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequenc ...[more]

PMID: 32327500

Similar Datasets

Project description:The 3' untranslated region (3'UTR) of mRNA plays several important roles in post-transcriptional gene regulation. Some of its functions include regulating mRNA stability by polyadenylation and microRNAs. However, the overall function of the Gata1 3’UTR in mammals has not been defined. In this study, we used CRISPR/CAS9 technology to knock out the sequence of the mouse Gata1 3’UTR. We found a defect in erythropoiesis in mutant mice, evidenced by macrocytic anemia at the baseline. Ablation of Gata1 3’UTR also resulted in a reduced number of erythroid precursors that might be associated with the cell cycle, especially G2/M disruption in fetal livers. Mechanistically, deletion of the Gata1 3’UTR destabilizes the Gata1 mRNA and ultimately reduces Gata1 protein levels. The low stability of the Gata1 mRNA is unlikely to be caused by the loss of binding of microRNAs or lack of polyadenylation; rather, in part, by the dissociation of AU-rich elements in the 3’UTR with a trans-activating factor Elavl1. Specifically, we transcribed Gata1 3’UTR in vitro and performed an RNA pulldown assay followed by mass spectrometry to profile the proteins that bind the 3’UTR. Gene Ontology analysis demonstrated that several proteins specifically targeting the 3’UTR were found to potentially bind Gata1 3’UTR, among which Elavl1 was in almost all categories related to mRNA stabilization. Western blotting and RNA immunoprecipitation confirmed the direct interaction of Gata1 3’UTR with Elavl1. Manipulation of Elavl1 activity and protein levels by the small molecule inhibitor Dihydro-tanshinone-I and Elavl1 overexpression in fetal liver erythroblasts confirmed Elavl1 as a stabilizing factor for Gata1 mRNA. Our findings shed light on the functional significance of the 3’UTR of Gata1 mRNA in the context of erythroid development. More importantly, our findings highlight the complexity and diversity of regulatory mechanisms that govern Gata1 mRNA stability and precise expression at the post-transcriptional levels. In addition, our findings prove that miRNAs are not always essential in vivo for controlling transcription factor levels to maintain body homeostasis.