Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

Identification of transcription termination defects at DNA hypomethylated transcription termination sites in DNA methyltransferase 3a-deficient vertebrates.

CpG methylation in genomic DNA is well known as a repressive epigenetic marker in eukaryotic transcription, and DNA methylation of the promoter regions is correlated with silencing of gene expression. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study has revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions (including transcription termination sites (TTSs)) during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we employed two strategies: informatic analysis using already deposited datasets of Dnmt3a-/- mouse cells and the zebrafish model system. Bioinformatic analysis using methylome and transcriptome data showed that hypomethylated differentially methylated regions overlapping the TTSs were associated with increased read counts and chimeric transcripts downstream of TTSs in Dnmt3a-/- Agouti-related protein neurons, but not in Dnmt3a-/- ES cells and MEFs. We experimentally detected increased read-through and chimeric transcripts downstream of hypomethylated TTSs in zebrafish maternal-zygotic dnmt3aa-/- mutants. This study is the first to identify transcription termination defects in DNA hypomethylated TTSs in Dnmt3a-/- vertebrates.

Computational analysis of sense-antisense chimeric transcripts reveals their potential regulatory features and the landscape of expression in human cells

Many human genes are transcribed from both strands and produce sense-antisense gene pairs. Sense-antisense (SAS) chimeric transcripts are produced upon the coalescing of exons/introns from both sense and antisense transcripts of the same gene. SAS chimera was first reported in prostate cancer cells. Subsequently, numerous SAS chimeras have been reported in the ChiTaRS-2.1 database. However, the landscape of their expression in human cells and functional aspects are still unknown. We found that longer palindromic sequences are a unique feature of SAS chimeras. Structural analysis indicates that a long hairpin-like structure formed by many consecutive Watson-Crick base pairs appears because of these long palindromic sequences, which possibly play a similar role as double-stranded RNA (dsRNA), interfering with gene expression. RNA–RNA interaction analysis suggested that SAS chimeras could significantly interact with their parental mRNAs, indicating their potential regulatory features. Here, 267 SAS chimeras were mapped in RNA-seq data from 16 healthy human tissues, revealing their expression in normal cells. Evolutionary analysis suggested the positive selection favoring sense-antisense fusions that significantly impacted the evolution of their function and structure. Overall, our study provides detailed insight into the expression landscape of SAS chimeras in human cells and identifies potential regulatory features.

The pluripotent stem cell-specific transcript ESRG is dispensable for human pluripotency

Human pluripotent stem cells (PSCs) express human endogenous retrovirus type-H (HERV-H), which exists as more than a thousand copies on the human genome and frequently produces chimeric transcripts as long-non-coding RNAs (lncRNAs) fused with downstream neighbor genes. Previous studies showed that HERV-H expression is required for the maintenance of PSC identity, and aberrant HERV-H expression attenuates neural differentiation potentials, however, little is known about the actual of function of HERV-H. In this study, we focused on ESRG, which is known as a PSC-related HERV-H-driven lncRNA. The global transcriptome data of various tissues and cell lines and quantitative expression analysis of PSCs showed that ESRG expression is much higher than other HERV-Hs and tightly silenced after differentiation. However, the loss of function by the complete excision of the entire ESRG gene body using a CRISPR/Cas9 platform revealed that ESRG is dispensable for the maintenance of the primed and naïve pluripotent states. The loss of ESRG hardly affected the global gene expression of PSCs or the differentiation potential toward trilineage. Differentiated cells derived from ESRG-deficient PSCs retained the potential to be reprogrammed into induced PSCs (iPSCs) by the forced expression of OCT3/4, SOX2, and KLF4. In conclusion, ESRG is dispensable for the maintenance and recapturing of human pluripotency.

Clinical, Histological, and Molecular Features of Solitary Fibrous Tumor of Bone: A Single Institution Retrospective Review

Primary solitary fibrous tumor (SFT) of the bone is extremely rare, with only few cases reported in the literature. We retrieved all cases of primary SFT of the bone treated at our institution and we assessed the morphology and the immunohistochemical and molecular features to investigate the clinical outcome of primary SFT of the bone and any clinical relevance of clinical and histological criteria of aggressiveness currently adopted for the soft tissues counterpart. Morphologically, 15 cases evidenced high cellularity, cytologic atypia, and foci of necrosis and were associated with more than 4 mitotic figures/10 HPF. Immunohistochemical analysis showed an expression of CD34 and of STAT6 immunopositivity in 95% and in 100% of cases, respectively. The presence of NAB2-STAT6 chimeric transcripts was found in 10 out of 12 cases in which RT-PCR analysis was feasible, whereas TERT promoter mutations analysis was feasible in 16 cases and only a C-to-T substitution in a heterozygous state was found in one DNA sample for the C228T genetic variant. P53 variants were assessed in 12 cases: 11 (91.6%) cases showed a variation, while in one case, no alteration was found. Disease-specific survival was 64% at 5 years and 49% at 10 years. Statistical analysis showed no correlation between survival and all the clinicopathological and molecular parameters evaluated. In conclusion, at difference to SFT of soft tissues, aggressive behavior of primary SFT of the bone seems to be independent from mitotic count or any other clinicopathological and molecular features.

LINE-1 and Alu Promote Breast Tumor Progression Via Forming Chimeric Transcripts

Background：LINE-1 (L1) and Alu were reported to regulate tumor development and progression by constituting chimeric transcripts with diverse sequences. The landscape of L1 or Alu chimeric transcripts in breast tumor has not been reported yet, so breast tumor L1 and Alu chimeric transcripts were investigated in this study by analyzing more than 50 billion RNA sequences of breast tumor tissues and adjacent normal tissues from TCGA database. Results：The expression of L1 and Alu in breast tumor tissues were significantly higher than that in the non-tumor tissues. High expression of L1 and Alu in breast tumor predicted poor prognosis. Further exploration demonstrated that L1 and Alu were extensively chimerically expressed with adjacent transcripts. 651 L1-mRNA chimeric transcripts and 1525 Alu-mRNA chimeric transcripts showed significantly different frequencies between non-tumor and tumor tissues. 1009 L1-lncRNA chimeric transcripts and 2575 Alu-lncRNA chimeric transcripts had significantly different frequencies between non-tumor and tumor tissues. Function cluster analysis demonstrated that these differently chimerically expressed genes were involved in multi facets of tumorigenesis, including metabolism, signal transduction, immune reaction, cell cycle and apoptosis, etc. Conclusions：Chimeric expression with different sequences might be an important mechanism for L1 and Alu to promote breast tumor progression.

Nsd2 Represses Endogenous Retrovirus MERVL in Embryonic Stem Cells

The facilitates chromatin transcription (FACT) complex is a histone H2A/H2B chaperone, which represses endogenous retroviruses (ERVs) and transcription of ERV-chimeric transcripts. It binds to both transcription start site and gene body region. Here, we investigated the downstream targets of FACT complex to identify the potential regulators of MERVL, which is a key 2-cell marker gene. H3K36me2 profile was positively correlated with that of FACT component Ssrp1. Among H3K36me2 deposition enzymes, Nsd2 was downregulated after the loss of Ssrp1. Furthermore, we demonstrated that Nsd2 repressed the expression of ERVs without affecting the expression of pluripotency genes. The expression of MERVL and 2-cell genes was partially rescued by Nsd2 overexpression. The enrichment of H3K36me2 decreased on MERVL-chimeric gene in ESCs without Ssrp1. Our study discovers that Nsd2 is a repressor of MERVL, and FACT partially represses MERVL expression by regulating the expression of Nsd2 and its downstream H3K36me2.

SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.