SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

RTCpredictor: identification of read-through chimeric RNAs from RNA sequencing data.

Read-through chimeric RNAs are being recognized as a means to expand the functional transcriptome and contribute to cancer tumorigenesis when mis-regulated. However, current software tools often fail to predict them. We have developed RTCpredictor, utilizing a fast ripgrep tool to search for all possible exon-exon combinations of parental gene pairs. We also added exonic variants allowing searches containing common SNPs. To our knowledge, it is the first read-through chimeric RNA specific prediction method that also provides breakpoint coordinates. Compared with 10 other popular tools, RTCpredictor achieved high sensitivity on a simulated and three real datasets. In addition, RTCpredictor has less memory requirements and faster execution time, making it ideal for applying on large datasets.

Evolutionary impact of chimeric RNAs on generating phenotypic plasticity in human cells.

Chimeric RNAs are generated by the fusion of the exons or introns of two genes. The generation of chimeric RNAs is important for the functional expansion of cells. Here, we describe the functional implications of chimeric RNAs for generating phenotypic plasticity from an evolutionary perspective.

Recent advances in cancer fusion transcript detection.

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts.

Potential multi-modal effects of provirus integration on HIV-1 persistence: lessons from other viruses.

Despite antiretroviral therapy (ART), HIV-1 persists as proviruses integrated into the genomic DNA of CD4+ T cells. The mechanisms underlying the persistence and clonal expansion of these cells remain incompletely understood. Cases have been described in which proviral integration can alter host gene expression to drive cellular proliferation. Here, we review observations from other genome-integrating human viruses to propose additional putative modalities by which HIV-1 integration may alter cellular function to favor persistence, such as by altering susceptibility to cytotoxicity in virus-expressing cells. We propose that signals implicating such mechanisms may have been masked thus far by the preponderance of defective and/or nonreactivatable HIV-1 proviruses, but could be revealed by focusing on the integration sites of intact proviruses with expression potential.

Comprehensive characterization of somatic mutations associated with chimeric RNAs in human cancers.

Chimeric RNAs, which can arise from gene recombination at the DNA level or non-canonical splicing events at the RNA level, have been identified as important roles in human tumors. Dysregulated gene expression caused by somatic mutations and altered splicing patterns of oncogenes or tumor suppressor genes can contribute to the development of tumors. Therefore, investigating the formation mechanism of chimeric RNAs via somatic mutations is critical for understanding tumor pathogenesis. This project is the first to propose studying the association between somatic single nucleotide variants and chimeric RNAs, identifying around 2900 somatic SNVs affecting chimeric RNAs in pan-cancer level. The somatic SNVs on chimeric RNAs were commonly observed in various types of tumor tissues, providing a valuable resource for future study. Additionally, these SNVs show distinct tumor specificity, and those with high frequency had a significant impact on the survival time of patients with tumors. Further research revealed that somatic SNVs associated with chimeric RNA (chiR-SNVs) were typically found within 10 nt of the junction site of chimeric RNAs and had a particularly significant effect on chimeric RNAs from different chromosomes. The enrichment analysis revealed that chiR-SNVs were significantly overrepresented in oncogenes and genes related to RNA binding proteins involved in RNA splicing, which could imply that chiR-SNVs may disrupt the process of RNA splicing and induce the occurrence of chimeric RNAs. This study sheds light on the potential molecular interaction mechanism between somatic SNVs and chimeric RNAs, which opens up a new avenue for researching disease pathway and tumorigenesis development.

SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data.

MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that function in post-transcriptional gene regulation through imperfect base pairing with mRNA targets which results in inhibition of translation and typically destabilization of bound transcripts. Sequence-based algorithms historically used to predict miRNA targets face inherent challenges in reliably reflecting in vivo interactions. Recent strategies have directly profiled miRNA-target interactions by crosslinking and ligation of sncRNAs to their targets within the RNA-induced silencing complex (RISC), followed by high throughput sequencing of the chimeric sncRNA:target RNAs. Despite the strength of these direct profiling approaches, standardized pipelines for effectively analyzing the resulting chimeric sncRNA:target RNA sequencing data are not readily available. Here we present SCRAP, a robust Small Chimeric RNA Analysis Pipeline for the bioinformatic processing of chimeric sncRNA:target RNA sequencing data. SCRAP consists of two parts, each of which are specifically optimized for the distinctive characteristics of chimeric small RNA sequencing reads: first, read processing and alignment and second, peak calling and annotation. We apply SCRAP to benchmark chimeric sncRNA:target RNA sequencing datasets generated by distinct molecular approaches, and compare SCRAP to existing chimeric RNA analysis pipelines. SCRAP has minimal hardware requirements, is cross-platform, and contains extensive annotation to broaden accessibility for processing small chimeric RNA sequencing data and enable insights about the targets of small non-coding RNAs in regulating diverse biological systems.

Identification of circular RNAs hosted by the RPGR ORF15 genomic locus.

Mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRorf15) cause X-linked retinitis pigmentosa, a severe and early onset inherited retinal degeneration. The underlying pathogenic mechanisms and variability in disease severity remain to be fully elucidated. The present study examines structural features of the ORF15 exonic region to provide new insights into the disease pathogenesis. Using canine and human RNA samples, we identified several novel RPGR ORF15-like linear RNA transcripts containing cryptic introns (exitrons) within the annotated exon ORF15. Furthermore, using outward-facing primers designed inside exitrons in the ORF15 exonic region, we found many of previously unidentified circular RNAs (circRNAs) that formed via back fusion of linear parts of the RPGRorf15 pre-mRNAs. These circRNAs (resistant to RNAse R treatment) were found in all studied cells and tissues. Notably, some circRNAs were present in cytoplasmic and polysomal RNA fractions. Although certain RPGR circRNAs may be cell type specific, we found some of the same circRNAs expressed in different cell types, suggesting similarities in their biogenesis and functions. Sequence analysis of RPGR circRNAs revealed several remarkable features, including identification of N6-methyladenosine (m6A) consensus sequence motifs and high prevalence of predictive microRNA binding sites pointing to the functional roles of these circRNAs. Our findings also illustrate the presence of non-canonical RPGR circRNA biogenesis pathways independent of the known back splicing mechanism. The obtained data on novel RPGR circRNAs further underline structural complexity of the RPGR ORF15 region and provide a potential molecular basis for the disease phenotypic heterogeneity.

Integration of SARS-CoV-2 RNA in infected human cells by retrotransposons: an unlikely hypothesis and old viral relationships.

Zhang et al. (Proc Natl Acad Sci 118:e2105968118, 2021) recently reported that SARS-CoV-2 RNA can be retrotranscribed and integrated into the DNA of human cells by the L1 retrotransposon machinery. This phenomenon could cause persistence of viral sequences in patients and may explain the prolonged PCR-positivity of SARS-CoV-2 infected patients, even long after the phase of active virus replication has ended. This commentary does critically review the available data on this topic and discusses them in the context of findings made for other exogenous viruses and ancestral endogenous retroviral elements.

Comparative study of bioinformatic tools for the identification of chimeric RNAs from RNA Sequencing.

Chimeric RNAs are gaining more and more attention as they have broad implications in both cancer and normal physiology. To date, over 40 chimeric RNA prediction methods have been developed to facilitate their identification from RNA sequencing data. However, a limited number of studies have been conducted to compare the performance of these tools; additionally, previous studies have become outdated as more software tools have been developed within the last three years. In this study, we benchmarked 16 chimeric RNA prediction software, including seven top performers in previous benchmarking studies, and nine that were recently developed. We used two simulated and two real RNA-Seq datasets, compared the 16 tools for their sensitivity, positive prediction value (PPV), F-measure, and also documented the computational requirements (time and memory). We noticed that none of the tools are inclusive, and their performance varies depending on the dataset and objects. To increase the detection of true positive events, we also evaluated the pair-wise combination of these methods to suggest the best combination for sensitivity and F-measure. In addition, we compared the performance of the tools for the identification of three classes (read-through, inter-chromosomal and intra-others) of chimeric RNAs. Finally, we performed TOPSIS analyses and ranked the weighted performance of the 16 tools.

RNA-mediated gene fusion in mammalian cells.

One of the hallmarks of cancer is the formation of oncogenic fusion genes as a result of chromosomal translocations. Fusion genes are presumed to form before fusion RNA expression. However, studies have reported the presence of fusion RNAs in individuals who were negative for chromosomal translocations. These observations give rise to "the cart before the horse" hypothesis, in which the genesis of a fusion RNA precedes the fusion gene. The fusion RNA then guides the genomic rearrangements that ultimately result in a gene fusion. However, RNA-mediated genomic rearrangements in mammalian cells have never been demonstrated. Here we provide evidence that expression of a chimeric RNA drives formation of a specified gene fusion via genomic rearrangement in mammalian cells. The process is: (i) specified by the sequence of chimeric RNA involved, (ii) facilitated by physiological hormone levels, (iii) permissible regardless of intrachromosomal (TMPRSS2-ERG) or interchromosomal (TMPRSS2-ETV1) fusion, and (iv) can occur in normal cells before malignant transformation. We demonstrate that, contrary to "the cart before the horse" model, it is the antisense rather than sense chimeric RNAs that effectively drive gene fusion, and that this disparity can be explained by transcriptional conflict. Furthermore, we identified an endogenous RNA AZI1 that functions as the "initiator" RNA to induce TMPRSS2-ERG fusion. RNA-driven gene fusion demonstrated in this report provides important insight in early disease mechanisms, and could have fundamental implications in the biology of mammalian genome stability, as well as gene-editing technology via mechanisms native to mammalian cells.

Interior circular RNA.

Formed by back splicing or back fusion of linear RNAs, circular RNAs (circRNAs) constitute a new class of non-coding RNAs of eukaryotes. Recent studies reveal a spliceosome-dependent biogenesis of circRNAs where circRNAs arise at the intron-exon junctions of mRNAs. In this study, using a novel de novo identification method, we show that circRNAs can originate from the interior regions of exons, introns, and intergenic transcripts in human, mouse and rice, which were referred to as interior circRNAs (i-circRNAs). Many i-circRNAs have some remarkable characteristics: multiple i-circRNAs may arise from the same genomic locus; their back fusion points may not be associated with the AG/GT splicing sites, but rather a new pair of motif AC/CT, their back fusion points are adjacent to complementary sequences; and they may circulate on short homologous sequences. We validated several i-circRNAs in HeLa cells by Polymerase Chain Reaction followed by Sanger sequencing. Our results combined showed that i-circRNAs are bona fide circRNAs, indicated novel biogenesis pathways independent of the splicing apparatus, and expanded our understanding of the origin, diversity, and complexity of circRNAs.

Functional heritage: the evolution of chimeric RNA into a gene.

Once believed to be unique features of neoplasia, chimeric RNAs are now being discovered in normal physiology. We speculated that some chimeric RNAs may be functional precursors of genes, and that forming chimeric RNA at the transcriptional level may be a 'trial' mechanism before the functional element is fixed into the genome. Supporting this idea, we identified a chimeric RNA, HNRNPA1L2-SUGT1 (H-S), whose sequence is highly similar to that of a 'pseudogene' MRPS31P5. Sequence analysis revealed that MRPS31P5 transcript is more similar to H-S chimeric RNA than its 'parent' gene, MRPS31. Evolutionarily, H-S precedes MRPS31P5, as it can be detected bioinformatically and experimentally in marmosets, which do not yet possess MRPS31P5 in their genome. Conversely, H-S is minimally expressed in humans, while instead, MRPS31P5 is abundantly expressed. Silencing H-S in marmoset cells resulted in similar phenotype as silencing MRPS31P5 in human cells. In addition, whole transcriptome analysis and candidate downstream target validation revealed common signalling pathways shared by the two transcripts. Interestingly, H-S failed to rescue the phenotype caused by silencing MPRS31P5 in human and rhesus cells, whereas MRPS31P5 can at least partially rescue the phenotype caused by silencing H-S in marmoset cells, suggesting that MRPS31P5 may have further evolved into a distinct entity. Thus, multiple lines of evidence support that MRPS31P5 is not truly a pseudogene of MRPS31, but a likely functional descendent of H-S chimera. Instead being a gene fusion product, H-S is a product of cis-splicing between adjacent genes, while MRPS31P5 is likely produced by genome rearrangement.

Activity and fidelity of human DNA polymerase α depend on primer structure.

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

PAX3-FOXO1 escapes miR-495 regulation during muscle differentiation.

Pax3 plays an essential role in myogenesis. Previously, we found a tumor-signature chimeric fusion RNA, PAX3-FOXO1 also present during muscle differentiation, raising the possibility of its physiological role. Here we demonstrated that the fusion is needed transiently for muscle lineage commitment. Interestingly, the fusion ortholog was not found in seven mouse muscle differentiation/regeneration systems, nor in other stem cell differentiation systems of another three mammal species. We noticed that Pax3 is expressed at a much lower level in human stem cells, and during muscle differentiation than in other mammals. Given the fact that the fusion and the parental Pax3 share common downstream targets, we reasoned that forming the fusion may be a mechanism for human cells to escape certain microRNA regulation on Pax3. By sequence comparison, we identified 16 candidate microRNAs that may specifically target the human PAX3 3'UTR. We used a luciferase reporter assay, examined the microRNAs expression, and conducted mutagenesis on the reporters, as well as a CRISPR/Cas9 mediated editing on the endogenous allele. Finally, we identified miR-495 as a microRNA that specifically targets human PAX3. Examining several other fusion RNAs revealed that the human-specificity is not limited to PAX3-FOXO1. Based on these observations, we conclude that PAX3-FOXO1 fusion RNA is absent in mouse, or other mammals we tested, the fusion RNA is a mechanism to escape microRNA, miR-495 regulation in humans, and that it is not the only human-specific fusion RNA.

Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

It Is Imperative to Establish a Pellucid Definition of Chimeric RNA and to Clear Up a Lot of Confusion in the Relevant Research.

There have been tens of thousands of RNAs deposited in different databases that contain sequences of two genes and are coined chimeric RNAs, or chimeras. However, "chimeric RNA" has never been lucidly defined, partly because "gene" itself is still ill-defined and because the means of production for many RNAs is unclear. Since the number of putative chimeras is soaring, it is imperative to establish a pellucid definition for it, in order to differentiate chimeras from regular RNAs. Otherwise, not only will chimeric RNA studies be misled but also characterization of fusion genes and unannotated genes will be hindered. We propose that only those RNAs that are formed by joining two RNA transcripts together without a fusion gene as a genomic basis should be regarded as authentic chimeras, whereas those RNAs transcribed as, and cis-spliced from, single transcripts should not be deemed as chimeras. Many RNAs containing sequences of two neighboring genes may be transcribed via a readthrough mechanism, and thus are actually RNAs of unannotated genes or RNA variants of known genes, but not chimeras. In today's chimeric RNA research, there are still several key flaws, technical constraints and understudied tasks, which are also described in this perspective essay.

Small mitochondrial-targeted RNAs modulate endogenous mitochondrial protein expression in vivo.

Endogenous mitochondrial genes encode critical oxidative phosphorylation components and their mutation results in a set of disorders known collectively as mitochondrial encephalomyopathies. There is intensive interest in modulating mitochondrial function as organelle dysfunction has been associated with numerous disease states. Proteins encoded by the mitochondrial genome cannot be genetically manipulated by current techniques. Here we report the development of a mitochondrial-targeted RNA expression system (mtTRES) utilizing distinct non-coding leader sequences (NCLs) and enabling in vivo expression of small mitochondrial-targeted RNAs. mtTRES expressing small chimeric antisense RNAs was used as translational inhibitors (TLIs) to target endogenous mitochondrial protein expression in vivo. By utilizing chimeric antisense RNA we successfully modulate expression of two mitochondrially-encoded proteins, ATP6 and COXII, and demonstrate the utility of this system in vivo and in human cells. This technique has important and obvious research and clinical implications.

RNA fusion in human retinal development.

Chimeric RNAs have been found in both cancerous and healthy human cells. They have regulatory effects on human stem/progenitor cell differentiation, stemness maintenance, and central nervous system development. However, whether they are present in human retinal cells and their physiological functions in the retinal development remain unknown. Based on the human embryonic stem cell-derived retinal organoids (ROs) spanning from days 0 to 120, we present the expression atlas of chimeric RNAs throughout the developing ROs. We confirmed the existence of some common chimeric RNAs and also discovered many novel chimeric RNAs during retinal development. We focused on CTNNBIP1-CLSTN1 (CTCL) whose downregulation caused precocious neuronal differentiation and a marked reduction of neural progenitors in human cerebral organoids. CTCL is universally present in human retinas, ROs, and retinal cell lines, and its loss-of-function biases the progenitor cells toward retinal pigment epithelial cell fate at the expense of retinal cells. Together, this work provides a landscape of chimeric RNAs and reveals evidence for their critical role in human retinal development.

Genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by chimeric RNA sequencing.

Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of in vivo interactions by sequencing of the resulting chimeric RNAs. Modifications are incorporated to increase the capacity for processing low-abundance samples and permit cell-type-selective profiling of sncRNA:target RNA interactions, as demonstrated in mouse brain cortex. CIMERA-seq represents a cohesive and optimized method for unambiguously characterizing the in vivo network of sncRNA:target RNA interactions in numerous biological contexts and even subcellular fractions. Genome-wide and cell-type-selective CIMERA-seq enhances researchers' ability to study gene regulation by sncRNAs in diverse model systems and tissue types.