Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells

Wilton, Joana; Tellier, Michael; Nojima, Takayuki; Costa, Ângela M.; Oliveira, Maria José; Moreira, Alexandra

doi:10.1016/bs.mie.2021.04.004

Cited by 3 publications

(6 citation statements)

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“…To assess alterations in gene expression, 3’UTR-APA and IPA events upon macrophage polarization, we performed 3’RNA-Seq, which provides both quantification of gene expression based on RNA levels at the 3’ end and APA profiles ( 32 ) and chromatin-bound RNA-Seq (ChrRNA-Seq) to pinpoint those events that occur in nascent RNA ( 34 ) ( Figure 1B ). 3’RNA-Seq reveals that 3005 genes have significantly altered expression in M1 in comparison to M0 macrophages ( Figure 1C ).…”

Section: Resultsmentioning

confidence: 99%

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Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

et al. 2023

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IntroductionMacrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3’UTR (3’UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3’UTR-APA and IPA in primary human macrophages was unclear.MethodsIn this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3’RNA-Seq was performed to quantify gene expression and characterize new 3’UTR-APA and IPA mRNA isoforms.ResultsOur results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3’UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3’UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3’UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses.DiscussionOur results reveal new 3’UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for SRSF12 in pro-inflammatory macrophages, key cells in the tumor response.

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Section: Resultsmentioning

confidence: 99%

“…Library preparation for 3’ RNASeq from healthy blood donors was performed as ( 32 ). Briefly, for M0, M1 and co-cultured macrophage samples, polyA + mRNA was obtained from total RNA using Dynabeads mRNA purification kit (ThermoFisher), according to manufacturer’s protocol.…”

Section: Discussionmentioning

confidence: 99%

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

et al. 2023

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“…The copyright holder for this this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02. 24.529734 doi: bioRxiv preprint To assess alterations in differential gene expression, 3'UTR-APA and IPA events upon macrophage polarization, we performed 3'RNA-Seq, which provides both quantification of gene expression based on RNA levels at the 3' end and APA profiles 36 and also chromatin-bound RNA-Seq (ChrRNA-Seq) to pinpoint those events that occur in nascent RNA 38 (Figure 1B). 3' RNA-Seq results reveal that 3005 genes show a significantly altered expression in M1 in comparison to M0 macrophages (Figure 1C).…”

Section: Pro-inflammatory Polarization Alters Primary Human Macrophag...mentioning

confidence: 99%

“…Macrophages can be experimentally differentiated from quasi-naïve, differentiated and unpolarized cells into a spectrum of inflammatory profiles. We obtained primary human CD14 + monocytes isolated from peripheral blood mononuclear cells (PBMCs) of healthy blood donor buffy coats, differentiated them into macrophages in the presence of M-CSF (herein referred as M0) and polarized them towards pro-inflammatory conditions (M1) by incubation with LPS and IFN-γ, following a well-established methodology [35][36][37] represented in Figure 1A. M0 macrophages are predominantly round/ameboid-like, while M1 macrophages present a fibroblast-like morphology (Figure 1A).…”

Section: Pro-inflammatory Polarization Alters Primary Human Macrophag...mentioning

confidence: 99%

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Wilton

Mendonça

Pereira-Castro

et al. 2023

Preprint

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Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3prime UTR (3prime UTR-APA) and intronic polyadenylation (iPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3prime UTR-APA and iPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3prime RNA-Seq were performed to quantify gene expression and characterize new 3prime UTR-APA and iPA mRNA isoforms. Our results show that polarization of human macrophages from naive to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3prime UTR and in iPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and iPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3prime UTR-APA and iPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induce 3prime UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro-inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 3prime UTR-APA and iPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools.

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“…Instability and variations in the CPA complex assembly, as well as the stochastic nature of the cleavage process, result in APA through the selection of a particular one of multiple PASs [ 9 , 56 , 70 , 71 ]. Therefore, the length and composition of mRNA 3′ untranslated regions (UTRs) vary, resulting in different sets of targets for RNA-binding proteins and miRNAs, which finally define the mRNA transport, lifespan, and translation efficiency [ 71 , 72 , 73 ]. In addition, the length of the poly(A) tail is also crucial for the same aspects of mRNA biology [ 70 , 74 , 75 ].…”

Section: Introductionmentioning

confidence: 99%

Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells

Omelina

Letiagina

Boldyreva

et al. 2022

IJMS

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Compared to transcription initiation, much less is known about transcription termination. In particular, large-scale mutagenesis studies have, so far, primarily concentrated on promoter and enhancer, but not terminator sequences. Here, we used a massively parallel reporter assay (MPRA) to systematically analyze the influence of short (8 bp) sequence variants (mutations) located downstream of the polyadenylation signal (PAS) on the steady-state mRNA level of the upstream gene, employing an eGFP reporter and human HEK293T cells as a model system. In total, we evaluated 227,755 mutations located at different overlapping positions within +17..+56 bp downstream of the PAS for their ability to regulate the reporter gene expression. We found that the positions +17..+44 bp downstream of the PAS are more essential for gene upregulation than those located more distal to the PAS, and that the mutation sequences ensuring high levels of eGFP mRNA expression are extremely T-rich. Next, we validated the positive effect of a couple of mutations identified in the MPRA screening on the eGFP and luciferase protein expression. The most promising mutation increased the expression of the reporter proteins 13-fold and sevenfold on average in HEK293T and CHO cells, respectively. Overall, these findings might be useful for further improving the efficiency of production of therapeutic products, e.g., recombinant antibodies.

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Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells

Cited by 3 publications

References 35 publications

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells

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