Phenotypic polarization of macrophages is regulated by a milieu of cues in the local tissue microenvironment. Currently, little is known about how the intrinsic regulators modulate proinflammatory (M1) versus prohealing (M2) macrophages activation. Here, it is observed that insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 (IGF2BP2)-deleted macrophages exhibit enhanced M1 phenotype and promote dextran sulfate sodium induced colitis development. However, the IGF2BP2 −/− macrophages are refractory to interleukin-4 (IL-4) induced activation and alleviate cockroach extract induced pulmonary allergic inflammation. Molecular studies indicate that IGF2BP2 switches M1 macrophages to M2 activation by targeting tuberous sclerosis 1 via an N6-methyladenosine (m 6 A)-dependent manner. Additionally, it is also shown a signal transducer and activators of transcription 6 (STAT6)-high mobility group AT-hook 2-IGF2BP2-peroxisome proliferator activated receptor-axis involves in M2 macrophages differentiation. These findings highlight a key role of IGF2BP2 in regulation of macrophages activation and imply a potential therapeutic target of macrophages in the inflammatory diseases.

Background Aberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor CRKL (v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing. Methods We analyzed the expression level of CRKL in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples. Results The expression of CRKL was mostly up-regulated in stage I cervical cancer samples. Knock-down of CRKL led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples. Conclusions This is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation. Electronic supplementary material The online version of this article (10.1186/s12885-019-5671-8) contains supplementary material, which is available to authorized users.

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Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly (A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development-and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.

Contagious ecthyma is a highly contagious disease with worldwide distribution, which is caused by the Orf virus (ORFV) belonging to the Parapoxvirus. To study the alteration of host gene expression in response to ORFV infection at the transcriptional level, several young small-tailed Han sheep were inoculated with ORFV, and their oral mucosa tissue samples (T0, T3, T7 and T15) were collected on day 0, 3, 7 and 15 after ORFV infection respectively. RNA-seq transcriptome comparisons were performed, showing that 1928, 3219 and 2646 differentially expressed genes (DEGs) were identified among T3 vs. T0, T7 vs. T0, and T15 vs. T0 respectively. Gene Ontology (GO) analyses of the DEGs from these comparisons, revealed that ORFV might provoke vigorous immune response of the host cells during the early stage of infection. Moreover, GO and network analysis showed that positive and negative regulative mechanisms of apoptosis were integrated in the host cells through up or down-regulating the expression level of DEGs involved in apoptotic pathways, in order to reach a homeostasis of oral mucosa tissues during the exposure to ORFV infection. In conclusion, our study for the first time describes the direct effects of ORFV on the global host gene expression of its host using high-throughput RNA sequencing, which provides a resource for future characterizing the interaction mechanism between the mammalian host and ORFV.

Background Tristetraprolin (TTP) is an RNA binding protein that plays a critical role in regulating proinflammatory immune responses by destabilizing target mRNAs via binding to their AU-rich elements (AREs) in the 3′-UTRs of mRNAs. A recent CLIP-seq study revealed that TTP-binding sites are enriched in the intronic regions of RNA. TTP is also a nuclear protein that exhibits putative DNA-binding activity. These features suggested that TTP might regulate gene transcription and/or alternative splicing of pre-mRNAs in the absence of stimulation. Results To elucidate the regulatory pattern of TTP, we cloned and overexpressed the human TTP-encoding gene, ZFP36, in HeLa cells in the absence of inflammatory stimuli. The transcriptomes of the control and ZFP36- overexpressing cells were sequenced and subjected to analysis and validation. Upon ZFP36 overexpression, the expression of genes associated with innate immunity, including those in the type I interferon signaling pathway and viral response, were specifically upregulated, implying a transcriptional regulatory mechanism associated with the predicted DNA binding activity of TTP. TTP preferentially regulated the alternative splicing of genes involved in the positive regulation of the I-κB/NF-κB cascade and the TRIF-dependent toll-like receptor, MAPK, TNF, and T cell receptor signaling pathways. Conclusions Our findings indicated that TTP may regulate the immune response via the regulation of alternative splicing and potentially transcription, which greatly expands the current understanding of the mechanisms of TTP-mediated gene regulation. Electronic supplementary material The online version of this article (10.1186/s12865-019-0292-1) contains supplementary material, which is available to authorized users.

DEAD-box helicase 41 (DDX41) is an RNA helicase and accumulating evidence has suggested that DDX41 is involved in pre-mRNA splicing during tumor development. However, the role of DDX41 in tumorigenesis remains unclear. In order to determine the function of DDX41, the human DDX41 gene was cloned and overexpressed in HeLa cells. The present study demonstrated that DDX41 overexpression inhibited proliferation and promoted apoptosis in HeLa cells. RNA-sequencing analysis of the transcriptomes in overexpressed and normal control samples. DDX41 regulated 959 differentially expressed genes compared with control cells. Expression levels of certain oncogenes were also regulated by DDX41. DDX41 selectively regulated the alternative splicing of genes in cancer-associated pathways including the EGFR and FGFR signaling pathways. DDX41 selectively upregulated the expression levels of five antigen processing and presentation genes ( HSPA1A, HSPA1B, HSPA6, HLA-DMB and HLA-G ) and downregulated other immune-response genes in HeLa cells. Additionally, DDX41-regulated oncogenes and antigen processing and presentation genes were associated with patient survival rates. Moreover, DDX41 expression was associated with immune infiltration in cervical and endocervical squamous cancer. The present findings showed that DDX41 regulated the cancer cell transcriptome at both the transcriptional and alternative splicing levels. The DDX41 regulatory network predicted the biological function of DDX41 in suppressing tumor cell growth and regulating cancer immunity, which may be important for developing anticancer therapeutics.