A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach.

5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

U1 snRNP regulates chromatin retention of noncoding RNAs.

Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.

Phase separation of RNA-binding protein promotes polymerase binding and transcription.

An RNA-involved phase-separation model has been proposed for transcription control. However, the molecular links that connect RNA to the transcription machinery remain missing. Here we find that RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), some being colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

Clinical efficacy of chemotherapy in colorectal cancer patients over 80 years old.

PURPOSE: Colorectal cancer (CRC) is a common and aggressive gastrointestinal cancer, and the prognostic impact associated with chemotherapy in super elderly (over 80 years old) patients remains poorly defined. We aimed to define the effect of chemotherapy on the prognosis of patients with CRC over 80 years old. PATIENTS AND METHODS: A retrospective study including CRC patients over 80 years old was conducted. The patients were screened from the Surveillance Epidemiology and End Results (SEER) database from 2010 to 2015. Overall survival (OS) and cancer-specific survival (CSS) were applied as the primary and secondary outcome. Cox proportional hazards regression models were used to evaluate factors associated with OS and CSS. Survival curves of OS and CSS were estimated by Kaplan-Meier method and compared by log-rank test. RESULTS: In total, 14,748 CRC patients over 80 years old were included in this study. The median patient age was 85 (IQR: 82-87). All patients were divided into surgical group and non-surgical group. The OS and CSS of the surgical group were significantly better than those of the non-surgical group (P < 0.001). Chemotherapy can improve OS and CSS for patients with stage III and IV (P < 0.001) in surgical group. For the super elderly patients with CRC, chemotherapy significantly improved OS and CSS in all TNM stages in non-surgical group. CONCLUSION: For super elderly patients with colorectal cancer, tumor treatment should not be abandoned because of their age. It is necessary to carry out clinical trials in super elderly patients.

Localization of RNAs in the nucleus: cis- and trans- regulation.

The subcellular localization of RNAs correlates with their function and how they are regulated. Most protein-coding mRNAs are exported into the cytoplasm for protein synthesis, while some mRNA species, long noncoding RNAs, and some regulatory element-associated unstable transcripts tend to be retained in the nucleus, where they function as a regulatory unit and/or are regulated by nuclear surveillance pathways. While the mechanisms regulating mRNA export and localization have been well summarized, the mechanisms governing nuclear retention of RNAs, especially of noncoding RNAs, are seldomly reviewed. In this review, we summarize recent advances in the mechanistic study of RNA nuclear retention, especially for noncoding RNAs, from the angle of cis-acting elements embedded in RNA transcripts and their interaction with trans-acting factors. We also try to illustrate the general principles of RNA nuclear retention and we discuss potential areas for future investigation.

Insight into novel RNA-binding activities via large-scale analysis of lncRNA-bound proteome and IDH1-bound transcriptome.

RNA-binding proteins (RBPs) play pivotal roles in directing RNA fate and function. Yet the current annotation of RBPs is largely limited to proteins carrying known RNA-binding domains. To systematically reveal dynamic RNA-protein interactions, we surveyed the human proteome by a protein array-based approach and identified 671 proteins with RNA-binding activity. Among these proteins, 525 lack annotated RNA-binding domains and are enriched in transcriptional and epigenetic regulators, metabolic enzymes, and small GTPases. Using an improved CLIP (crosslinking and immunoprecipitation) method, we performed genome-wide target profiling of isocitrate dehydrogenase 1 (IDH1), a novel RBP. IDH1 binds to thousands of RNA transcripts with enriched functions in transcription and chromatin regulation, cell cycle and RNA processing. Purified IDH1, but not an oncogenic mutant, binds directly to GA- or AU-rich RNA that are also enriched in IDH1 CLIP targets. Our study provides useful resources of unconventional RBPs and IDH1-bound transcriptome, and convincingly illustrates, for the first time, the in vivo and in vitro RNA targets and binding preferences of IDH1, revealing an unanticipated complexity of RNA regulation in diverse cellular processes.

Clinical value of bedside abdominal sonography performed by certified sonographer in emergency evaluation of blunt abdominal trauma.

PURPOSE: To investigate the accuracy and efficiency of bedside ultrasonography application performed by certified sonographer in emergency patients with blunt abdominal trauma. METHODS: The study was carried out from 2017 to 2019. Findings in operations or on computed tomography (CT) were used as references to evaluate the accuracy of bedside abdominal ultrasonography. The time needed for bedside abdominal ultrasonography or CT examination was collected separately to evaluate the efficiency of bedside abdominal ultrasonography application. RESULTS: Bedside abdominal ultrasonography was performed in 106 patients with blunt abdominal trauma, of which 71 critical patients received surgery. The overall diagnostic accordance rate was 88.68%. The diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation, retroperitoneal hematoma and multiple abdominal organ injury were 100%, 94.73%, 94.12%, 20.00%, 100% and 81.48%, respectively. Among the 71 critical patients, the diagnostic accordance rate was 94.37%, in which the diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation and multiple abdominal organ injury were 100%, 100%, 100%, 20.00% and 100%. The mean time for imaging examination of bedside abdominal ultrasonography was longer than that for CT scan (4.45 ± 1.63 vs. 2.38 ± 1.19) min; however, the mean waiting time before examination (7.37 ± 2.01 vs. 16.42 ± 6.37) min, the time to make a diagnostic report (6.42 ± 3.35 vs. 36.26 ± 13.33) min, and the overall time (17.24 ± 2.33 vs. 55.06 ± 6.96) min were shorter for bedside abdominal ultrasonography than for CT scan. CONCLUSION: Bedside ultrasonography application provides both efficiency and reliability for the assessment of blunt abdominal trauma. Especially for patients with free peritoneal effusion and critical patients, bedside ultrasonography has been proved obvious advantageous. However, for negative bedside ultrasonography patients with blunt abdominal trauma, we recommend further abdominal CT scan or serial ultrasonography scans subsequently.

RNF8 is responsible for ATRA resistance in variant acute promyelocytic leukemia with GTF2I/RARA fusion, and inhibition of the ubiquitin-proteasome pathway contributes to the reversion of ATRA resistance.

BACKGROUND: GTF2I-RARA is a newly identified RARA fusion gene in variant acute promyelocytic leukemia (APL) patients with t(7;17)(q11;q21). Clinical manifestation in the patient showed that it is a sort of ATRA-insensitive oncogene and is different from the classic PML-RARA in terms of therapeutic reaction. METHODS: To reveal the functional characteristics and regulating mechanism of the GTF2I-RARA fusion gene, we established a GTF2I-RARA-transfected HL60 cell model and examined its sensitivity to ATRA by western blot, MTT assay, flow cytometry, and Wright-Giemsa staining. Coimmunoprecipitation and confocal microscopy were used to examine the binding of GTF2I-RARA and transcriptional corepressors. We also performed ChIP-seq to search for potential target genes. Immunoprecipitation, ubiquitination assay, western blot, luciferase assay, and real-time PCR were used to analyze the effects of RNF8 on RARA. Flow cytometry and Wright-Giemsa staining were used to study the effect of MG132 and ATRA on the GTF2I-RARA-transfected HL60 cell model. RESULT: We confirmed resistance of GTF2I-RARA to ATRA. Compared with PML-RARA, GTF2I-RARA has a higher affinity to HDAC3 under ATRA treatment. Using the ChIP-sequencing approach, we identified 221 GTF2I-RARA binding sites in model cells and found that the RING finger protein 8 (RNF8) is a target gene of GTF2I-RARA. RNF8 participates in disease progression and therapy resistance in APL with the GTF2I-RARA transcript. Elevated RNF8 expression promotes the interaction between RARA and RNF8 and induces RARA Lys-48 linkage ubiquitylation and degradation, resulting in attenuated transcriptional activation of RARA. CONCLUSION: Our results suggest that RNF8 is a key GTF2I-RARA downstream event. Using the combination of MG132 and ATRA to treat GTF2I-RARA-HL60 cells, a synergistic effect leading to GTF2I-RARA-HL60 cell differentiation was confirmed. Taken together, the targeting of RNF8 may be an alternative choice for treatment in variant APL with GTF2I-RARA fusion.

A lower dose of fluorescein sodium is more suitable for confocal laser endomicroscopy: a feasibility study.

BACKGROUND AND AIMS: Image quality can be guaranteed with the conventional dosage of fluorescein sodium in probe-based confocal laser endomicroscopy (pCLE). However, yellow discoloration of the skin seriously affects daily life and simultaneously increases the risk of adverse events such as allergic reactions. The aim of this study was to test whether a lower dosage of fluorescein sodium can provide satisfactory image quality and to compare the diagnostic accuracy of gastric intestinal metaplasia (GIM) through a randomized blind controlled trial. METHODS: Consecutive patients were randomly assigned to different doses of fluorescein sodium. Image quality was determined by the endoscopists' subjective assessments and signal-to-noise ratio (SNR) assessment systems. Skin discoloration was tested using a neonatal transcutaneous jaundice detector. In addition, consecutive patients with a known or suspected diagnosis of GIM were examined by pCLE with the lower dose and the traditional dose. RESULTS: Only 0.01 mL/kg dose of 10% fluorescein sodium led to a significant decrease in image quality (P < .05), and a dose of 0.02 mL/kg had the highest SNR value (P < .05). There were no significant differences in skin discoloration between the 0.01 mL/kg and 0.02 mL/kg doses (P = .148) and no statistical difference in the diagnostic accuracy of pCLE for GIM between the 0.02 mL/kg and 0.10 mL/kg doses (P > .05). The kappa values for the correlation between pCLE and histopathology were 0.867 (95% confidence interval, 0.782-0.952) and 0.891 (95% confidence interval, 0.811-0.971). CONCLUSIONS: The 0.02 mL/kg dose of 10% fluorescein sodium seems to be the best dose for pCLE in the upper GI tract, with comparable image quality with the conventional dose and insignificant skin discoloration. This dose is also very efficient for the diagnosis of GIM.

Two cases of atrial myxoma with calcification and ossification as the main features.

BACKGROUND: Cardiac myxomas are the most common type of primary cardiac tumors in adults, but they can have variable features that make them difficult to diagnose. We report two cases of atrial myxoma with calcification or ossification, which are rare pathological subgroups of myxoma. CASE PRESENTATION: A 47-year-old woman and a 35-year-old man presented to our hospital with different symptoms. Both patients had a history of chronic diseases. Transthoracic and transesophageal echocardiography revealed a mass in the left or right atrium, respectively, with strong echogenicity and echogenic shadows. The masses were suspected to be malignant tumors with calcification or ossification. Contrast transthoracic echocardiography(cTEE) showed low blood supply within the lesions. The patients underwent surgical resection of the atrial mass, and the pathology confirmed myxoma with partial ossification or massive calcification. CONCLUSION: We report two rare cases of atrial myxoma with calcification or ossification and analyze their ultrasonographic features. Transthoracic echocardiography and cTEE can provide valuable information for the diagnosis and management of such mass. However, distinguishing calcification and ossification in myxoma from calcification in malignant tumors is challenging. More studies are needed to understand the pathogenesis and imaging characteristics of these myxoma variants.

Chemoproteomic profiling unveils binding and functional diversity of endogenous proteins that interact with endogenous triplex DNA.

Triplex DNA structures, formed when a third DNA strand wraps around the major groove of DNA, are key molecular regulators and genomic threats. However, the regulatory network governing triplex DNA dynamics remains poorly understood. Here we reveal the binding and functional repertoire of proteins that interact with triplex DNA through chemoproteomic profiling in living cells. We develop a chemical probe that exhibits exceptional specificity towards triplex DNA. By employing a co-binding-mediated proximity capture strategy, we enrich triplex DNA interactome for quantitative proteomics analysis. This enables the identification of a comprehensive list of proteins that interact with triplex DNA, characterized by diverse binding properties and regulatory mechanisms in their native chromatin context. As a demonstration, we validate DDX3X as an ATP-independent triplex DNA helicase to unwind substrates with a 5' overhang to prevent DNA damage. Overall, our study provides a valuable resource for exploring the biology and translational potential of triplex DNA.

Crosstalk between epitranscriptomic and epigenomic modifications and its implication in human diseases.

Crosstalk between N6-methyladenosine (m6A) and epigenomes is crucial for gene regulation, but its regulatory directionality and disease significance remain unclear. Here, we utilize quantitative trait loci (QTLs) as genetic instruments to delineate directional maps of crosstalk between m6A and two epigenomic traits, DNA methylation (DNAme) and H3K27ac. We identify 47 m6A-to-H3K27ac and 4,733 m6A-to-DNAme and, in the reverse direction, 106 H3K27ac-to-m6A and 61,775 DNAme-to-m6A regulatory loci, with differential genomic location preference observed for different regulatory directions. Integrating these maps with complex diseases, we prioritize 20 genome-wide association study (GWAS) loci for neuroticism, depression, and narcolepsy in brain; 1,767 variants for asthma and expiratory flow traits in lung; and 249 for coronary artery disease, blood pressure, and pulse rate in muscle. This study establishes disease regulatory paths, such as rs3768410-DNAme-m6A-asthma and rs56104944-m6A-DNAme-hypertension, uncovering locus-specific crosstalk between m6A and epigenomic layers and offering insights into regulatory circuits underlying human diseases.

Noncoding RNA-chromatin association: Functions and mechanisms.

Pervasive transcription of the mammalian genome produces hundreds of thousands of noncoding RNAs (ncRNAs). Numerous studies have suggested that some of these ncRNAs regulate multiple cellular processes and play important roles in physiological and pathological processes. Notably, a large subset of ncRNAs is enriched on chromatin and participates in regulating gene expression and the dynamics of chromatin structure and status. In this review, we summarize recent advances in the functional study of chromatin-associated ncRNAs and mechanistic insights into how these ncRNAs associate with chromatin. We also discuss the potential future challenges which still need to be overcome in this field.

Circ_SEC61A1 contributes to the progression of multiple myeloma cells via regulating miR-660-5p/CDK6 axis.

BACKGROUND: Circular RNAs (circRNAs) have been reported to play critical roles in the malignant progression of diverse human cancers, including multiple myeloma (MM). This study aimed to explore the functional role and underlying mechanism of circ_SEC61A1 in MM progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect the expression levels of circ_SEC61A1, microRNA (miR)-660-5p and cyclin-dependent kinase 6 (CDK6) mRNA. The localization of circ_SEC61A1 in MM cells was tested by the subcellular fractionation location assay. Actinomycin D assay was conducted to determine the characteristics of circ_SEC61A1. Cell proliferation was evaluated by colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot assay was exploited to examine the expression of proteins. Cell migration and invasion were tested via transwell assay, and cell apoptosis was measured by flow cytometry analysis. Dual-luciferase reporter assay was utilized to confirm the interaction between miR-660-5p and circ_SEC61A1 or CDK6. RESULTS: Circ_SEC61A1 level was increased in MM tissues and cells. Circ_SEC61A1 was a stable circRNA and mainly located in cytoplasm. Circ_SEC61A1 silence restrained the proliferation, metastasis and expedited the apoptosis in MM cells. CDK6 was identified as the target of miR-660-5p, and circ_SEC61A1 sponged miR-660-5p to positively regulate CDK6 expression. The inhibitory impacts of circ_SEC61A1 knockdown on the progression of MM cells were mitigated by miR-660-5p inhibition. MiR-660-5p overexpression blocked the malignant phenotypes of MM cells by targeting CDK6. CONCLUSION: Our study manifested that circ_SEC61A1 could accelerate MM progression at least partially through modulating miR-660-5p/CDK6 axis.

Gene activation guided by nascent RNA-bound transcription factors.

Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.

Long non­coding RNA ANRIL is a potential indicator of disease progression and poor prognosis in acute myeloid leukemia.

The present study explored the association of long non­coding RNA (lncRNA) antisense non­coding RNA in the INK4 locus (ANRIL) with the development of acute myeloid leukemia (AML) clinical features and prognosis of patients with AML. Bone marrow mononuclear cells (BMMCs) were obtained from 178 patients with de novo AML prior to initial therapy and from 30 healthy donors. The expression of lncRNA ANRIL in BMMCs was detected by reverse transcription­quantitative PCR. Complete remission (CR) was assessed after induction therapy. Event­free survival (EFS) and overall survival (OS) were evaluated during the follow­up. The levels of lncRNA ANRIL were increased in patients with AML compared with those in healthy donors and were capable of distinguishing patients with AML from healthy donors (area under the curve, 0.886; 95% CI, 0.820­0.952). Furthermore, lncRNA ANRIL was associated with an increased occurrence internal tandem duplications in the FMS­like tyrosine kinase 3, decreased occurrence inv(16) or t(16;6), intermediate­risk and poor­risk stratification while no association of lncRNA ANRIL was identified with French­American­British classification, cytogenetics, isolated biallelic CCAAT/enhancer­binding protein α mutation and nucleophosmin 1 mutation in patients with AML. Furthermore, lncRNA ANRIL was significantly associated with a lower CR rate. In addition, EFS and OS were shorter in patients with high expression of lncRNA ANRIL compared with those in patients with low expression of lncRNA ANRIL. Multivariate Cox regression analyses revealed that high expression of lncRNA ANRIL, poor­risk stratification and white blood cells (>10.0x109 cells/l) were independent prognostic factors for shorter EFS, while high expression of lncRNA ANRIL and poorer risk stratification were independent prognostic factors for shorter OS. The present results suggested that lncRNA ANRIL has clinical relevance as a biomarker for assisting diagnosis treatment decisions and prognosis prediction and the identification of potential drug target for AML.