Abstract:Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNA interference screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eukaryotic translation initiation factor 4A2 (eIF4A2) and defined its mechanistic action through ribosomal protein S26–independent and –dependent ribosomes in translation… Show more
“…We validated these five candidates by repeating the shRNA experiment in another mouse ESC line, CCE, with the same protocol described (Figure 1A ), and confirmed that the pluripotency loss and the differentiation induction were reproducible and consistent with the target KD efficiency ( Supplementary Figure S2A, B ). Among these five candidates, Ddx2b ( 28 , 29 ), Ddx6 ( 30–33 ), Ddx18 ( 34 ) and Ddx47 ( 35 ) were previously reported to play important roles in ESCs, not only validating our screening results but also highlighting Ddx48 as a novel candidate. Examination of their expression levels among various tissues and cell lines revealed that, like Ddx18, Ddx48 is also particularly enriched in ESCs ( Supplementary Figure S3 ), indicating its ESC-specific roles.…”
Section: Resultssupporting
confidence: 85%
“…We also noticed that, upon eIF4A3 KD, the reduction of Cyclin B1 protein was more pronounced than that of eIF4A3 (Figure 6G , H ) and that the RPF reduction on Ccnb1 mRNA mainly happens at the translation initiation region (5′ UTR and the beginning of the coding region) (Figure 5D , shaded in purple, the RPF tracks). In ribosome profiling, it is well known that for the proteins bound at translation initiation regions, their expression changes can lead to RPF alteration mainly at these translation initiation regions, with much less or even no alteration on the rest of mRNA bodies, and such a reduction can dramatically decrease the protein levels of these mRNAs ( 29 ). This could apply to eIF4A3 KD-induced Ccnb1 change in our study, considering that eIF4A3 is mainly bound around the first exon–exon junction of Ccnb1 mRNA with the enrichment at the translation initiation region (Figure 5D ).…”
RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNA interference (RNAi) screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes the loss of pluripotency via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during the cell cycle progression of ESCs. Our results reveal a previously unappreciated role for eIF4A3 and its associated EJC in maintaining stem cell pluripotency through post-transcriptional control of the cell cycle.
“…We validated these five candidates by repeating the shRNA experiment in another mouse ESC line, CCE, with the same protocol described (Figure 1A ), and confirmed that the pluripotency loss and the differentiation induction were reproducible and consistent with the target KD efficiency ( Supplementary Figure S2A, B ). Among these five candidates, Ddx2b ( 28 , 29 ), Ddx6 ( 30–33 ), Ddx18 ( 34 ) and Ddx47 ( 35 ) were previously reported to play important roles in ESCs, not only validating our screening results but also highlighting Ddx48 as a novel candidate. Examination of their expression levels among various tissues and cell lines revealed that, like Ddx18, Ddx48 is also particularly enriched in ESCs ( Supplementary Figure S3 ), indicating its ESC-specific roles.…”
Section: Resultssupporting
confidence: 85%
“…We also noticed that, upon eIF4A3 KD, the reduction of Cyclin B1 protein was more pronounced than that of eIF4A3 (Figure 6G , H ) and that the RPF reduction on Ccnb1 mRNA mainly happens at the translation initiation region (5′ UTR and the beginning of the coding region) (Figure 5D , shaded in purple, the RPF tracks). In ribosome profiling, it is well known that for the proteins bound at translation initiation regions, their expression changes can lead to RPF alteration mainly at these translation initiation regions, with much less or even no alteration on the rest of mRNA bodies, and such a reduction can dramatically decrease the protein levels of these mRNAs ( 29 ). This could apply to eIF4A3 KD-induced Ccnb1 change in our study, considering that eIF4A3 is mainly bound around the first exon–exon junction of Ccnb1 mRNA with the enrichment at the translation initiation region (Figure 5D ).…”
RNA helicases are involved in multiple steps of RNA metabolism to direct their roles in gene expression, yet their functions in pluripotency control remain largely unexplored. Starting from an RNA interference (RNAi) screen of RNA helicases, we identified that eIF4A3, a DEAD-box (Ddx) helicase component of the exon junction complex (EJC), is essential for the maintenance of embryonic stem cells (ESCs). Mechanistically, we show that eIF4A3 post-transcriptionally controls the pluripotency-related cell cycle regulators and that its depletion causes the loss of pluripotency via cell cycle dysregulation. Specifically, eIF4A3 is required for the efficient nuclear export of Ccnb1 mRNA, which encodes Cyclin B1, a key component of the pluripotency-promoting pathway during the cell cycle progression of ESCs. Our results reveal a previously unappreciated role for eIF4A3 and its associated EJC in maintaining stem cell pluripotency through post-transcriptional control of the cell cycle.
“…We further detected 16 ZNF genes that exhibit a co-expression pattern with the histone genes in the specific stem cell and growth conditions (Figure 6B and C ), of which seven genes were closely located within chromosome 19 (13q42-13q43) (Figure 6D ). Previous studies reported the connection between stem cell identity and histones or zinc fingers: the activation of multiple histone variants, including H1, H2A and H3, in maintaining ESC pluripotency and ESC fate ( 103 , 104 ), the role of histone variant H3.3 on the maintenance of HSC stemness ( 105 ), and co-localized 31 zinc-finger genes within topologically associating domains on chr19 in human ESCs ( 106 ). We found a higher co-expression pattern between histones and co-localized zinc fingers in ESCs in xenogenic medium and HSCs in L-GLN medium (Figure 6A and C ).…”
Several factors, including tissue origins and culture conditions, affect the gene expression of undifferentiated stem cells. However, understanding the basic identity across different stem cells has not been pursued well despite its importance in stem cell biology. Thus, we aimed to rank the relative importance of multiple factors to gene expression profile among undifferentiated human stem cells by analyzing publicly available RNA-seq datasets. We first conducted batch effect correction to avoid undefined variance in the dataset as possible. Then, we highlighted the relative impact of biological and technical factors among undifferentiated stem cell types: a more influence on tissue origins in induced pluripotent stem cells than in other stem cell types; a stronger impact of culture condition in embryonic stem cells and somatic stem cell types, including mesenchymal stem cells and hematopoietic stem cells. In addition, we found that a characteristic gene module, enriched in histones, exhibits higher expression across different stem cell types that were annotated by specific culture conditions. This tendency was also observed in mouse stem cell RNA-seq data. Our findings would help to obtain general insights into stem cell quality, such as the balance of differentiation potentials that undifferentiated stem cells possess.
“…Tubulin alpha chain and beta chains, Vimentin as well as Eif4a2 and Pdia6 (Supplementary Data 8 ). These two factors are known to be involved in stem cell pluripotency [ 48 ] and metastasis [ 49 ]. Fig.…”
Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.
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