Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3′UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3′UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages.

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The RNA-binding protein QKI5 is a direct target of C/EBPα and delays macrophage differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0412 ◽

2012 ◽

Vol 23 (9) ◽

pp. 1628-1635 ◽

Cited By ~ 18

Author(s):

Haiyan Fu ◽

Guodong Yang ◽

Mengying Wei ◽

Li Liu ◽

Liang Jin ◽

...

Keyword(s):

Molecular Mechanisms ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Macrophage Differentiation ◽

Endogenous Expression ◽

Enhancer Binding Protein ◽

Reporter Assays ◽

Direct Target ◽

Site Blocking

Differentiated macrophages are essential for the innate immune system; however, the molecular mechanisms underlying the generation of macrophages remain largely unknown. Here we show that the RNA-binding protein QKI, mainly QKI-5, is transcriptionally activated in the early differentiated monocytic progenitors when CCAAT/enhancer-binding protein (C/EBP) α is expressed. The forced expression of C/EBPα increases the endogenous expression of QKI. Chromatin immunoprecipitation analysis and reporter assays further confirm that C/EBPα activates the transcription of QKI, primarily by binding to the distal C/EBPα-binding site. Blocking the induction of QKI using RNA interference enhances the expression of endogenous CSF1R and facilitates macrophage differentiation. Further study of the mechanism reveals that QKI-5 facilitates the degradation of CSF1R mRNA by interacting with the distal QRE in the 3′ untranslated region. In summary, we show that in committed macrophage progenitors, C/EBPα-activated QKI-5 negatively regulates macrophage differentiation by down-regulating CSF1R expression, forming a negative feedback loop during macrophage differentiation.

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C/EBPα acts as an RNA binding protein essential for its chromatin recruitment and promotes Macrophage differentiation

10.1101/2021.10.20.465166 ◽

2021 ◽

Author(s):

Yanzhou Zhang ◽

Mahmoud Bassal ◽

Daniel Friedrich ◽

Simone Ummarino ◽

Tom Verbiest ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Specific Gene ◽

Macrophage Differentiation ◽

Binding Domains ◽

Rna Binding Domains ◽

Neutrophil Differentiation

C/EBPα has known to be a transcription factor that involved in Neutrophil differentiation for decades. However, exploring the Chromatin RNA Immunoprecipitation Sequencing (RIP), we discover that C/EBPα is a RNA binding protein mainly interacts with RNA introns. Structure study and RNA electrophoretic mobility shift assay (REMSA) show that C/EBPα interacts with RNA through two novel RNA binding domains distinct from its DNA binding domain. Mouse bone marrow transplantation and in vitro cytokine assay reveal that C/EBPα RNA binding is critical for Macrophage differentiation but not Neutrophil differentiation. Mechanically, RNA binding domains control specific gene transcription. In particular, PU.1 intron 4 RNA interacts with C/EBPα and recruit C/EBPα to its enhancer site, which facilitate PU.1 expression. Taken together, C/EBPα is demonstrated to be a RNA binding protein with unique function distinct from its DNA binding activity. Our finding transforms our knowledge of transcriptional regulation by transcription factor.

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RNA-Binding Profiles of CKAP4 as an RNA-Binding Protein in Myocardial Tissues

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.773573 ◽

2021 ◽

Vol 8 ◽

Author(s):

Hong Zhu ◽

Yanfeng Zhang ◽

Chengliang Zhang ◽

Zhongshang Xie

Keyword(s):

Cardiac Remodeling ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Axonal Guidance ◽

Signal Pathways ◽

Protein Markers ◽

Cell Cycle Protein ◽

Protein Ubiquitination ◽

Atrial Muscle

Background: Pathological tissue remodeling such as fibrosis is developed in various cardiac diseases. As one of cardiac activated-myofibroblast protein markers, CKAP4 may be involved in this process and the mechanisms have not been explored.Methods: We assumed that CKAP4 held a role in the regulation of cardiac fibrotic remodeling as an RNA-binding protein. Using improved RNA immunoprecipitation and sequencing (iRIP-seq), we sought to analyze the RNAs bound by CKAP4 in normal atrial muscle (IP1 group) and remodeling fibrotic atrial muscle (IP2 group) from patients with cardiac valvular disease. Quantitative PCR and Western blotting were applied to identify CKAP4 mRNA and protein expression levels in human right atrium samples.Results: iRIP-seq was successfully performed, CKAP4-bound RNAs were characterized. By statistically analyzing the distribution of binding peaks in various regions on the reference human genome, we found that the reads of IP samples were mainly distributed in the intergenic and intron regions implying that CKAP4 is more inclined to combine non-coding RNAs. There were 913 overlapping binding peaks between the IP1 and IP2 groups. The top five binding motifs were obtained by HOMER, in which GGGAU was the binding sequence that appeared simultaneously in both IP groups. Binding peak-related gene cluster enrichment analysis demonstrated these genes were mainly involved in biological processes such as signal transduction, protein phosphorylation, axonal guidance, and cell connection. The signal pathways ranking most varied in the IP2 group compared to the IP1 group were relating to mitotic cell cycle, protein ubiquitination and nerve growth factor receptors. More impressively, peak analysis revealed the lncRNA-binding features of CKAP4 in both IP groups. Furthermore, qPCR verified CKAP4 differentially bound lncRNAs including LINC00504, FLJ22447, RP11-326N17.2, and HELLPAR in remodeling myocardial tissues when compared with normal myocardial tissues. Finally, the expression of CKAP4 is down-regulated in human remodeling fibrotic atrium.Conclusions: We reveal certain RNA-binding features of CKAP4 suggesting a relevant role as an unconventional RNA-binding protein in cardiac remodeling process. Deeper structural and functional analysis will be helpful to enrich the regulatory network of cardiac remodeling and to identify potential therapeutic targets.

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