HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells.

BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

Distinct binding pattern of EZH2 and JARID2 on RNAs and DNAs in hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is one of the most malignant cancers worldwide, with high mortality. However, the molecular regulatory mechanisms of liver cancer, especially transcriptional and post-transcriptional mechanisms, should be further studied. Here we used chromatin and cross-linking immunoprecipitation with high throughput sequencing methods (ChIP-seq and CLIP-seq) to capture the global binding profiles on RNAs and DNAs of Enhancer of zeste homolog 2 (EZH2) and its partner Jumonji And AT-Rich Interaction Domain Containing 2 (JARID2) in liver carcinoma cell lines (HepG2) and normal liver cell line (THLE-2), respectively. We also integrated HCC transcriptome data from the TCGA to analyze the expression pattern of bound genes. We found that EZH2 and JARID2 both showed distinct binding profiles between HepG2 and THLE-2 cells. By binding to the primary RNAs, bound transcripts of EZH2 and JARID2 in HepG2 showed significantly increased transcriptional levels in HCC patients. By performing gene set enrichment analysis (GSEA), the bound transcripts were also highly related to HCC development. We also found EZH2 and JARID2 could specifically bind to several long noncoding RNAs (lncRNAs), including H19. By exploring the DNA binding profile, we detected a dramatically repressed DNA binding ability of EZH2 in HepG2 cells. We also found that the EZH2-bound genes showed slightly increased transcriptional levels in HepG2 cells. Integrating analysis of the RNA and DNA binding profiles suggests EZH2 and JARID2 shift their binding ability from DNA to RNA in HepG2 cells to promote cancer development in HCC. Our study provided a comprehensive and distinct binding profile on RNAs and DNAs of EZH2 and JARID2 in liver cancer cell lines, suggesting their potential novel functional manners to promote HCC development.

Two novel RNA-binding proteins identification through computational prediction and experimental validation.

Since RBPs play important roles in the cell, it's particularly important to find new RBPs. We performed iRIP-seq and CLIP-seq to verify two proteins, CLIP1 and DMD, predicted by RBPPred whether are RBPs or not. The experimental results confirm that these two proteins have RNA-binding activity. We identified significantly enriched binding motifs UGGGGAGG, CUUCCG and CCCGU for CLIP1 (iRIP-seq), DMD (iRIP-seq) and DMD (CLIP-seq), respectively. The computational KEGG and GO analysis show that the CLIP1 and DMD share some biological processes and functions. Besides, we found that the SNPs between DMD and its RNA partners may be associated with Becker muscular dystrophy, Duchenne muscular dystrophy, Dilated cardiomyopathy 3B and Cardiovascular phenotype. Among the thirteen cancers data, CLIP1 and another 300 oncogenes always co-occur, and 123 of these 300 genes interact with CLIP1. These cancers may be associated with the mutations occurred in both CLIP1 and the genes it interacts with.

Analysis of Bulk RNA Sequencing Data Reveals Novel Transcription Factors Associated With Immune Infiltration Among Multiple Cancers.

Tumor-infiltrating immune cells shape the tumor microenvironment and are closely related to clinical outcomes. Several transcription factors (TFs) have also been reported to regulate the antitumor activity and immune cell infiltration. This study aimed to quantify the populations of different immune cells infiltrated in tumor samples based on the bulk RNA sequencing data obtained from 50 cancer patients using the CIBERSORT and the EPIC algorithm. Weighted gene coexpression network analysis (WGCNA) identified eigengene modules strongly associated with tumorigenesis and the activation of CD4+ memory T cells, dendritic cells, and macrophages. TF genes FOXM1, MYBL2, TAL1, and ERG are central in the subnetworks of the eigengene modules associated with immune-related genes. The analysis of The Cancer Genome Atlas (TCGA) cancer data confirmed these findings and further showed that the expression of these potential TF genes regulating immune infiltration, and the immune-related genes that they regulated, was associated with the survival of patients within multiple cancers. Exome-seq was performed on 24 paired samples that also had RNA-seq data. The expression quantitative trait loci (eQTL) analysis showed that mutations were significantly more frequent in the regions flanking the TF genes compared with those of non-TF genes, suggesting a driver role of these TF genes regulating immune infiltration. Taken together, this study presented a practical method for identifying genes that regulate immune infiltration. These genes could be potential biomarkers for cancer prognosis and possible therapeutic targets.

The m6A Reader IGF2BP2 Regulates Macrophage Phenotypic Activation and Inflammatory Diseases by Stabilizing TSC1 and PPARγ.

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 (m6A)-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-γ 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.

SUVA: splicing site usage variation analysis from RNA-seq data reveals highly conserved complex splicing biomarkers in liver cancer.

Most of the current alternative splicing (AS) analysis tools are powerless to analyse complex splicing. To address this, we developed SUVA (Splice sites Usage Variation Analysis) that decomposes complex splicing events into five types of splice junction pairs. By analysing real and simulated data, SUVA showed higher sensitivity and accuracy in detecting AS events than the compared methods. Notably, SUVA detected extensive complex AS events and screened out 69 highly conserved and dominant AS events associated with cancer. The cancer-associated complex AS events in FN1 and the co-regulated RNA-binding proteins were significantly correlated with patient survival.

DDX41 regulates the expression and alternative splicing of genes involved in tumorigenesis and immune response.

DEAD­box helicase 41 (DDX41) is an RNA helicase and accumulating evidence has suggested that DDX41 is involved in pre­mRNA 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. RNA­sequencing 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 cancer­associated pathways including the EGFR and FGFR signaling pathways. DDX41 selectively upregulated the expression levels of five antigen processing and presentation genes (HSPA1A, HSPA1B, HSPA6, HLA­DMB and HLA­G) and downregulated other immune­response genes in HeLa cells. Additionally, DDX41­regulated 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.

TCTP participates in hepatic metabolism by regulating gene expression involved in insulin resistance.

Translationally controlled tumor protein (TCTP) has various cellular functions and molecular interactions, many related to its growth-promoting and antiapoptotic properties. Recently, TCTP expression was reported to increases in insulin-resistant mice fed with high-fat diet. TCTP is a multifunctional protein, but its role in liver metabolism is unclear. Here, we investigated the function and mechanism of TCTP in HepG2 cells. Knock-down of TCTP led to 287 differentially expressed genes (DEGs) that were highly associated with cellular apoptosis and signal response, TNF and NF-κB signaling pathways, glycolysis/gluconeogenesis, insulin resistance, FoxO and insulin signaling pathways, adipocytokine and AMPK signaling pathways. shTCTP downregulated the expression of the key gluconeogenesis enzyme phosphoenolpyruvate carboxykinase (PCK1). Furthermore, TCTP regulated the alternative splicing of genes enriched in the phospholipid biosynthetic process and glycerophospholipid metabolism. We further showed that shTCTP down-regulated the intracellular levels of triglyceride and total cholesterol. Our results showed that TCTP regulates the liver cell transcriptome at both the transcriptional and alternative splicing levels. The TCTP regulatory network predicts the biological functions of TCTP in glucose and lipid metabolism, and also insulin resistance, which may be associated with liver metabolism and diseases such as nonalcoholic fatty liver disease.

Genome-wide identification of AGO18b-bound miRNAs and phasiRNAs in maize by cRIP-seq.

BACKGROUND: Argonaute proteins (AGOs) are important players in the regulation of plant development by directing sRNAs to target mRNAs. In maize (Zea mays), AGO18b is a tassel-enriched and grass-specific AGO. Previous studies have shown that AGO18b is highly expressed in tassels during meiosis and negatively regulates determinacy of spikelet meristems. However, binding profile on RNAs and acting mechanisms of AGO18b remain unknown. RESULTS: In this study, we explored the binding profile of AGO18b in maize tassel by UV cross-linking RNA immunoprecipitation, followed by deep sequencing of these cDNA libraries (cRIP-seq), and systematically studied AGO18b-associated small RNAs and mRNAs by bioinformatics analysis. By globally analyzing the phased small-interfering RNA (phasiRNA) and miRNA abundance bound by AGO18b, we found AGO18b primarily binds to 21-nt phasiRNAs/miRNAs with a 5'-uridine and binds less strongly to 24-nt phasiRNAs with a 5'-adenosine in the premeiotic tassels. The abundance profile of AGO18b-associated miRNAs was different from their expression profile. Moreover, AGO18b strongly binds to miR166a-3p. We then obtained the AGO18b-bound mRNA targets of miR166a-3p by cRIP-seq, and confirmed the molecular function of AGO18b in regulating spikelet meristems. CONCLUSIONS: Our results indicated that AGO18b binds to phasiRNAs with obvious 5 prime end bias under different sRNA length. MiRNAs and their target mRNAs associated with AGO18b indicated the molecular mechanisms of AGO18b as a negative regulator of inflorescence meristem and tassel development through integrating both phasiRNAs and miRNA pathways, which extended our view of sRNA regulation in flower development and provided potential methods to control pollination in the future.

Hfq Globally Binds and Destabilizes sRNAs and mRNAs in Yersinia pestis.

Hfq is a ubiquitous Sm-like RNA-binding protein in bacteria involved in physiological fitness and pathogenesis, while its in vivo binding nature remains elusive. Here we reported genome-wide Hfq-bound RNAs in Yersinia pestis, a causative agent of plague, by using cross-linking immunoprecipitation coupled with deep sequencing (CLIP-seq) approach. We show that the Hfq binding density is enriched in more than 80% mRNAs of Y. pestis and that Hfq also globally binds noncoding small RNAs (sRNAs) encoded by the intergenic, antisense, and 3' regions of mRNAs. An Hfq U-rich stretch is highly enriched in sRNAs, while motifs partially complementary to AGAAUAA and GGGGAUUA are enriched in both mRNAs and sRNAs. Hfq-binding motifs are enriched at both terminal sites and in the gene body of mRNAs. Surprisingly, a large fraction of the sRNA and mRNA regions bound by Hfq and those downstream are destabilized, likely via a 5'P-activated RNase E degradation pathway, which is consistent with a model in which Hfq facilitates sRNA-mRNA base pairing and the coupled degradation in Y. pestis These results together have presented a high-quality Hfq-RNA interaction map in Y. pestis, which should be important for further deciphering the regulatory role of Hfq-sRNAs in Y. pestis IMPORTANCE Discovered in 1968 as an Escherichia coli host factor that was essential for replication of the bacteriophage Qß, the Hfq protein is a ubiquitous and highly abundant RNA-binding protein in many bacteria. With the assistance of Hfq, small RNAs in bacteria play important roles in regulating the stability and translation of mRNAs by base pairing. In this study, we want to elucidate the Hfq-assisted sRNA-mRNA regulation in Yersinia pestis A global map of Hfq interaction sites in Y. pestis was obtained by sequencing cDNAs converted from the Hfq-bound RNA fragments using UV cross-linking coupled immunoprecipitation technology. We demonstrate that Hfq could bind to hundreds of sRNAs and the majority of mRNAs in Y. pestis The enriched binding motifs in sRNAs and mRNAs are complementary to each other, suggesting a general base-pairing mechanism for sRNA-mRNA interaction. The Hfq-bound sRNA and mRNA regions were both destabilized. The results suggest that Hfq binding facilitates sRNA-mRNA base pairing and coordinates their degradation, which might enable Hfq to surveil the homeostasis of most mRNAs in bacteria.