What is Chimeric RNA?
Chimeric RNA, also known as fusion RNA, refers to RNA molecules that are formed as a result of the fusion of two or more different RNA transcripts. Chimeric RNA, a unique class of RNA molecules formed through various molecular mechanisms, such as trans-splicing, read-through transcription, and fusion genes. These hybrid transcripts play a significant role in diverse biological processes and have gained substantial attention in recent years.
Chimeric RNA Formation
Like mentioned earlier, the formation of chimeric RNAs involves multiple mechanisms. Chimeric RNA refers to a type of RNA molecule that is formed by the fusion of genetic material from two or more distinct genes. These chimeras can arise through different mechanisms, including chromosomal translocation, cis-splicing, or trans-splicing.
- Trans-splicing occurs when exons from different genes are spliced together, generating novel RNA molecules. Read-through transcription refers to the transcriptional read-through of adjacent genes, leading to the production of fusion transcripts. Fusion genes, resulting from chromosomal rearrangements, create chimeric RNA molecules by merging portions of two separate genes. These mechanisms give rise to unique transcripts that can have functional implications, such as altered protein-coding potential, regulatory effects, or generation of novel biomarkers.
- In chromosomal translocation, portions of two separate chromosomes break and rejoin, resulting in the fusion of two different genes. This rearrangement can occur within a single cell, leading to the production of chimeric RNA molecules.
- Cis splicing involves the abnormal joining of exons from two different genes located on the same chromosome. This can occur due to genetic mutations or errors in the splicing process. Trans-splicing, on the other hand, involves the joining of exons from different genes that are located on different chromosomes. This process requires the action of special trans-splicing machinery.
Chimeric RNAs (Mukherjee et al., 2022)
Chimeric RNAs have been observed in various organisms, including humans, and have been found to play a role in a variety of diseases, particularly cancer. They can contribute to tumorigenesis by generating abnormal proteins or disrupting normal cellular processes. Chimeric RNAs have also been implicated in other diseases, such as neurological disorders.
Sequencing Technologies for Chimeric RNA Detection
Chimeric RNA molecules, which arise from genomic rearrangements, alternative splicing, or fusion events, have emerged as crucial players in diverse biological processes and disease pathogenesis. Traditional sequencing methods, and next-generation sequencing (NGS) technologies as well as emerging long-read sequencing platforms, have traditionally been employed for chimeric RNA detection.
- Traditional sequencing methods, like Sanger sequencing and reverse transcription PCR (RT-PCR), have been historically employed for chimeric RNA detection. However, these techniques are limited in their ability to detect and characterize complex chimeric events and lack scalability.
- Next-generation sequencing technologies, particularly RNA-seq, have revolutionized chimeric RNA research. RNA-seq enables the high-throughput sequencing of transcriptomes, facilitating the discovery of novel chimeric events. It can efficiently identify multiple chimeric transcripts and DNA structural variants, especially when coupled with long RNA-seq reads and true chimeric mRNAs. RNA-seq provides valuable insights into transcriptome complexity and offers opportunities to study alternative splicing events and isoform diversity. Additionally, single-cell RNA-seq techniques enable the detection and analysis of chimeric RNA at the single-cell level, uncovering insights into cellular heterogeneity and disease processes.
- Emerging sequencing technologies, such as nanopore sequencing and PacBio sequencing, offer long-read sequencing capabilities. These technologies can provide valuable information about full-length chimeric transcripts, uncovering complex structural variations and alternative splicing events. But they have their own advantages and features. Nanopore sequencing offers the advantage of producing long reads, which are essential for capturing full-length chimeric transcripts, which provide information on the sequence and structural characteristics of chimeric RNA molecules, including complex structural variations and alternative splicing events. Furthermore, it enables the detection of base modifications and RNA modifications, contributing to the understanding of post-transcriptional RNA processing. PacBio sequencing, also known as Single Molecule, Real-Time (SMRT) sequencing, represents another long-read sequencing technology that holds promise for chimeric RNA analysis. This technology employs circular consensus sequencing (CCS) to generate highly accurate long reads. It offers high-resolution insights into RNA molecules and facilitates the investigation of intricate transcriptome landscapes.
ChimPipe method for detection of fusion genes and transcription-induced chimeras from RNA-seq data. (Rodríguez-Martí et al., 2017)
Computational Analysis of Chimeric RNA Data
Chimeric RNA data analysis necessitates a multifaceted computational framework to unravel the intricacies inherent in these composite transcripts.
In general, analyzing chimeric RNA data involves several computational steps:
A. Preprocessing and quality control. Preprocessing chimeric RNA data mandates meticulous artifact filtering and curation of low-quality reads to ensure the integrity and reliability of subsequent analyses. By applying sophisticated techniques, such as noise reduction algorithms, sequence trimming, and adapter removal, spurious artifacts are mitigated, fostering robust downstream analysis.
B. Alignment and mapping. The alignment and mapping phase entails the meticulous alignment of sequencing reads to reference genomes or transcriptomes, thereby enabling the discernment of chimeric RNA junctions. This process necessitates the utilization of sophisticated alignment algorithms, including splice-aware aligners or de novo assembly methods, to accurately identify and characterize these fusion events.
C. Identification and quantification of chimeric RNA. It necessitates the deployment of specialized algorithms capable of deciphering the presence and relative abundance of chimeric transcripts. Advanced techniques, such as fusion gene detection algorithms, breakpoint analysis, or statistical modeling approaches, empower researchers to discern intricate patterns and uncover novel chimeric events lurking within the dataset.
D. Visualization and interpretation. Complex chimeric RNA structures present an intellectual challenge that demands state-of-the-art visualization and interpretation tools. These tools facilitate the comprehension of intricate chimeric RNA arrangements by offering visual representations, such as circular plots, heatmaps, or interactive networks. Integration with complementary datasets, such as gene expression profiles or functional annotations, enriches the interpretative capacity, unraveling potential functional roles and underlying mechanisms of chimeric RNA molecules.
Fusion Finding and de novo Assembly Tools For chimeric RNA Sequencing
There are various fusion finding and de novo assembly programs that offer a range of methods and algorithms to detect fusion events and reconstruct transcriptomes from RNA-Seq data. Researchers can choose the appropriate tool based on their specific requirements, data characteristics, and the type of analysis they wish to perform.
| Fusion Finding Algorithms |
| BreakDancer |
Designed to detect genomic structural variations, including gene fusions, using paired-end sequencing data. It identifies and characterizes fusion events by analyzing discordant read pairs and split reads. |
| FusionSeq |
A computational pipeline for detecting fusion transcripts from RNA-Seq data. It integrates multiple signals, including read alignments, read counts, and spanning distances, to identify fusion events. |
| MapSplice |
A splice junction mapper for RNA-Seq data that can also detect fusion transcripts. It aligns reads to the genome and identifies fusion junctions based on the alignment patterns. |
| Tophat-fusion |
A component of the Tophat software suite for RNA-Seq analysis. It detects fusion transcripts by mapping reads to the reference genome and searching for novel junctions between different genes. |
| deFuse |
A computational tool that identifies fusion transcripts using paired-end RNA-Seq data. It employs a statistical framework to identify fusion events based on the alignment patterns of the paired-end reads. |
| FusionHunter |
Designed to detect fusion transcripts by aligning RNA-Seq reads to the genome. It applies filtering criteria to distinguish true fusion events from mapping artifacts and background noise. |
| SnowShoes-FTD |
Snowshoes-FTD (Finding Transcripts with Deletions) identifies fusion transcripts in RNA-Seq data by aligning reads to the reference genome and searching for breakpoints that indicate fusion events. |
| ChimeraScan |
A fusion detection tool that uses a combination of paired-end and split-read alignments to identify fusion events. It provides statistical measures to assess the confidence of detected fusion candidates. |
| FusionMap |
Designed to detect fusion transcripts using RNA-Seq data. It aligns reads to the genome and identifies fusion candidates based on spanning reads and split reads that support fusion junction. |
| FusionFinder |
Designed for detecting fusion transcripts in pediatric cancers. It utilizes paired-end RNA-seq data and employs a two-step approach that first identifies candidate fusion junctions and then validates them using statistical filtering. |
| FusionAnalyser |
A comprehensive tool for the identification and characterization of fusion transcripts from RNA-Seq data. It integrates read alignment, clustering, and fusion scoring algorithms to detect and prioritize fusion events. |
| SAOPfusion |
A fusion detection program that utilizes SOAP (Short Oligonucleotide Alignment Program) for read mapping and fusion identification. It considers both paired-end and split-read information to detect fusion events. |
| SAOPfuse |
Another fusion detection tool based on the SOAP algorithm. It identifies fusion transcripts by analyzing paired-end reads and split reads to identify fusion junctions between genes. |
| FusionCatcher |
A fusion gene detection tool that combines different algorithms for detecting fusion transcripts from RNA-Seq data. It uses both read mapping and de novo assembly approaches to identify fusion events. |
| ViralFusionSeq |
Designed for detecting fusion transcripts involving viral sequences. It identifies fusion events by aligning reads to both the human reference genome and viral genomes, allowing the detection of virus-host fusion transcripts. |
| PRADA |
A comprehensive analysis tool for RNA-Seq data. It includes a module for detecting fusion transcripts by aligning reads to the genome and identifying fusion junctions. |
| Chimera |
A R/Bioconductor package for the detection and analysis of fusion transcripts from RNA-Seq data. It utilizes multiple alignment strategies and statistical models to identify fusion events. |
| TRUP |
A tool for de novo assembly of RNA-Seq data, which can also detect fusion transcripts. It leverages long-read sequencing data to reconstruct full-length transcripts and identify fusion events. |
| De novo Assembly Algorithms |
| EBARDenovo |
A de novo assembly tool specifically designed for RNA-Seq data. It constructs transcript isoforms by assembling overlapping reads and inferring alternative splicing events. |
| Trinity |
De novo transcriptome assembly tool for RNA-Seq data. It employs a three-step approach that combines read clustering, de Bruijn graph construction, and transcript reconstruction to generate comprehensive transcriptome assemblies. |
| Trans-ABySS |
Utilizes a parallelized algorithm to construct transcript isoforms from RNA-Seq data. It handles alternative splicing and fusion events and generates comprehensive transcriptome assemblies. |
| Oases |
Specifically designed to handle RNA-Seq data. It uses a Velvet-based approach and implements a de Bruijn graph algorithm to assemble transcripts, including alternative isoforms. |
Chimeric RNA Sequencing Advances Detection and Research
Structural Variations and Chimeric RNA Formation
The identification and characterization of chimeric RNAs arising from structural variations, such as chromosomal rearrangements and translocations, require comprehensive genomic analyses. Employing state-of-the-art sequencing techniques, such as whole transcriptome sequencing, NGS or long-read RNA sequencing, to precisely delineate breakpoints and decipher the underlying mechanisms leading to chimeric RNA formation. By integrating various computational algorithms and advanced data visualization methods, meticulously annotate these structural variations and dissect their impact on the transcriptome can be achieved.
Transcriptomic Landscape and Functional Consequences
To comprehend the global landscape of chimeric RNAs and their functional implications, conducting large-scale transcriptomic profiling using high-throughput RNA sequencing is important. This involves generating high-quality, strand-specific RNA-Seq libraries, followed by specialized bioinformatics pipelines for accurate fusion transcript detection, abundance estimation, and differential expression analysis. By integrating these findings with other genomic data, such as chromatin accessibility or DNA methylation profiles, I unravel the regulatory networks and cellular pathways influenced by chimeric RNA molecules.
You can read our article Detecting Fusion Transcripts by RNA Sequencing in Screening Tumors for more knowledge about fusion transcripts in tumors.
Long Non-coding RNA (lncRNA) Chimeras
Investigate the formation, diversity, and functional roles of chimeric transcripts originating from non-coding regions of the genome is a part of exploring the emerging field of lncRNA chimeras. Employing cutting-edge sequencing technologies, including total RNA-Seq and Cap Analysis of Gene Expression (CAGE), the intricate interplay between lncRNAs and coding genes through chimeric RNA generation can be studied. By deciphering the mechanisms governing their biogenesis and unraveling their regulatory functions, it may contribute to our understanding of the non-coding RNA landscape and its involvement in complex biological processes.
Single-Cell Chimeric RNA Analysis
Single-cell RNA sequencing (scRNA-Seq) has revolutionized our ability to study cellular heterogeneity and identify rare cell populations. Leveraging this technology, I investigate the presence and dynamics of chimeric RNAs at the single-cell level. By combining scRNA-Seq with sophisticated computational algorithms and statistical frameworks, the cellular contexts and spatiotemporal dynamics of chimeric RNA expression can be unraveled. This enables us to discern their contribution to cellular diversity, developmental processes, and disease progression.
Functional Characterization and Mechanistic Insights
Beyond the identification and annotation of chimeric RNAs, uncovering their functional consequences and mechanistic underpinnings is also important. This involves employing advanced molecular biology techniques, such as CRISPR-based gene editing or RNA interference (RNAi), to manipulate chimeric RNA expression in cellular models. By integrating functional assays, transcriptomic analyses, and network-based approaches, the impact of chimeric RNAs on cellular processes, molecular interactions, and disease phenotypes can be elucidated.
Applications of Chimeric RNA Research
Chimeric RNA, a fascinating area of research, has emerged as a promising avenue with immense potential in diverse fields. This unique class often resulting from gene fusion events or other structural rearrangements by using chimeric RNA sequencing technologies, has been found to hold significant implications in multiple domains.
In the realm of cancer research, chimeric RNA molecules have demonstrated their value as diagnostic and prognostic biomarkers. By providing insights into tumor heterogeneity, chimeric RNA enables researchers to unravel the complex landscape of tumors, leading to a deeper understanding of their subtypes and the identification of potential therapeutic targets. Furthermore, the analysis of chimeric RNA can be leveraged to monitor treatment response and detect minimal residual disease, facilitating personalized and targeted therapies.
Moving into the realm of developmental biology, chimeric RNA has proven to be intimately involved in embryonic development, tissue differentiation, and organogenesis. By studying chimeric RNA, researchers gain a unique vantage point to delve into the intricate processes that shape an organism's development. This knowledge not only contributes to our fundamental understanding of developmental biology but also offers insights into developmental disorders and potential avenues for intervention.
In the field of neurological disorders, chimeric RNA emerges as a key player in unraveling the complex mechanisms underlying conditions such as neurodegenerative diseases and psychiatric disorders. Alterations in chimeric RNA expression and fusion events have been associated with these disorders, presenting new opportunities for biomarker discovery, disease classification, and potential therapeutic targets. The investigation of chimeric RNA promises to deepen our understanding of the intricate molecular landscape of neurological disorders.
Lastly, chimeric RNA research holds great promise in infectious diseases. By shedding light on host-pathogen interactions, immune responses, and potential therapeutic targets, chimeric RNA analysis provides valuable insights into the molecular mechanisms employed by pathogens. This knowledge not only aids in unraveling the intricate dynamics of infectious diseases but also facilitates the development of innovative strategies for targeted therapeutics and intervention.
In summary, chimeric RNA research represents a burgeoning field that holds immense potential across various disciplines. From its diagnostic and prognostic applications in cancer research to its contributions in developmental biology, neurological disorders, and infectious diseases, chimeric RNA research offers unprecedented opportunities to uncover the intricacies of biological processes and disease mechanisms. With continued exploration and advancements, chimeric RNA holds the promise of revolutionizing our understanding of diseases and opening new doors for effective diagnostics and therapeutics in the future.
References:
- Mukherjee, Sumit, and Milana Frenkel-Morgenstern. "Evolutionary impact of chimeric RNAs on generating phenotypic plasticity in human cells." Trends in Genetics 38.1 (2022): 4-7.
- Rodríguez-Martí, Bernardo, et al. "ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data." BMC genomics 18.1 (2017): 1-17.
