GO:0160091 spliceosome-depend formation of circular RNA: Back-Splicing Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0160091 describes the spliceosome-dependent back-splicing process that generates circular RNAs (circRNAs) from pre-mRNAs.
• Back-splicing joins a downstream splice donor to an upstream splice acceptor, forming a covalently closed loop.
• CircRNAs are abundant in the brain and other tissues, and many are conserved across species.
• CircRNAs can act as miRNA sponges, protein scaffolds, and templates for translation.
• Dysregulated circRNA biogenesis is linked to cancer, neurodegeneration, and cardiovascular disease.
• CRISPR knockout, point mutation, knock-in, and overexpression models enable functional dissection of circRNA biogenesis.
Description
Circular RNAs (circRNAs) are a large class of covalently closed, single-stranded RNA molecules generated by a process termed back-splicing. The Gene Ontology term GO:0160091, spliceosome-depend formation of circular RNA, defines the formation of circRNAs by back-splicing circularization of pre-mRNAs in a spliceosome-dependent process. This process is distinct from canonical linear splicing because it joins a downstream splice donor to an upstream splice acceptor, producing a circular rather than linear RNA product. Since the discovery that circRNAs are abundant and tissue-specific, particularly in the human fetal brain, they have emerged as key regulators of gene expression and potential biomarkers. Understanding GO:0160091 is therefore essential for researchers studying RNA processing, neural development, and disease mechanisms. The spliceosome, a large ribonucleoprotein complex, catalyzes both canonical and back-splicing, and its components are central to circRNA formation. Recent studies have revealed that circRNA biogenesis is regulated by cis-acting elements and trans-acting factors, and that circRNAs can be translated, exported, and degraded through specific pathways. This article provides a research-grade overview of GO:0160091, covering its definition, mechanism, key genes, disease relevance, and experimental methods for functional studies.
spliceosome-depend formation of circular RNA At A Glance
| GO ID | GO:0160091 |
|---|---|
| GO term | spliceosome-depend formation of circular RNA |
| Ontology | biological_process |
| Synonym | None |
| Definition | Formation of circular RNAs (circRNAs) by back-splicing circularization of pre-mRNAs in a spliceosome-dependent process. |
| Major function | Generation of covalently closed circular RNA molecules from pre-mRNAs via back-splicing. |
| Cellular location | Nucleus, particularly at splice sites of pre-mRNAs. |
| Key molecular players | Spliceosome components, RNA-binding proteins, and cis-acting intronic elements. |
| Related processes | Canonical splicing, alternative splicing, RNA export, and translation. |
What Is GO:0160091?
GO:0160091, spliceosome-depend formation of circular RNA, is a biological process defined as the formation of circular RNAs (circRNAs) by back-splicing circularization of pre-mRNAs in a spliceosome-dependent process. In this process, the spliceosome catalyzes a non-canonical splicing reaction that links a downstream 5' splice site to an upstream 3' splice site, generating a circular RNA molecule. This definition excludes circRNA formation pathways that do not require the spliceosome, such as those mediated by group I introns or other ribozymes.
Why Is spliceosome-depend formation of circular RNA Important in Cell Biology?
GO:0160091 is important because circRNAs generated by this process are abundant, evolutionarily conserved, and functionally diverse molecules that regulate gene expression at multiple levels. CircRNAs can act as microRNA sponges, protein scaffolds, and even templates for translation, and they are resistant to exonucleases due to their closed-loop structure. Dysregulation of circRNA biogenesis has been implicated in cancer, neurodegenerative disorders, and cardiovascular diseases, making the spliceosome-dependent formation of circRNAs a critical area of research. Moreover, engineered circRNAs are being developed as stable therapeutic agents, highlighting the translational potential of understanding this process.
• CircRNAs are highly enriched in the brain and are involved in neural development and synaptic function.
• Back-splicing generates circRNAs that can regulate gene expression by sponging miRNAs and proteins.
• CircRNAs are stable due to their covalently closed structure, making them attractive biomarkers and therapeutics.
• Dysregulated circRNA biogenesis is associated with multiple cancers, including hepatocellular carcinoma and glioblastoma.
• CircRNAs can be translated into proteins, expanding the proteome diversity.
• The spliceosome-dependent mechanism is conserved across eukaryotes, enabling model organism studies.
• CircRNA export from the nucleus is a regulated process that impacts their cellular functions.
• Understanding GO:0160091 aids in designing synthetic circRNAs for therapeutic applications.
• CircRNAs are potential biomarkers for disease diagnosis and prognosis.
• CRISPR-based editing of circRNA biogenesis pathways can reveal causal roles in disease.
What Happens During spliceosome-depend formation of circular RNA?
Recognition of Back-Splicing Signals
In simple terms: The cell identifies specific sequences in the pre-mRNA that tell it to make a circle instead of a line.
Back-splicing is guided by cis-acting elements, such as complementary sequences in flanking introns (e.g., Alu elements) that bring the splice sites into proximity. The spliceosome recognizes the downstream 5' splice site and the upstream 3' splice site, which are normally used for linear splicing, but in back-splicing they are joined in reverse order. This recognition is facilitated by RNA-binding proteins that stabilize the interaction between the splice sites.
Spliceosome Assembly and Catalysis
In simple terms: A large molecular machine called the spliceosome assembles on the RNA and cuts and joins the ends to form a circle.
The spliceosome, composed of small nuclear ribonucleoproteins (snRNPs) and associated proteins, assembles on the pre-mRNA in a stepwise manner. The catalytic core of the spliceosome performs two transesterification reactions that ligate the downstream 5' splice site to the upstream 3' splice site, resulting in a circular RNA. This process is ATP-dependent and requires the activity of DEAD-box helicases.
Formation of the Circular RNA Product
In simple terms: The ends are sealed together, creating a stable loop that is resistant to degradation.
The back-splicing reaction produces a covalently closed circular RNA molecule with a unique back-splice junction (BSJ). The BSJ is a signature feature used for circRNA detection and quantification. The circular RNA is then released from the spliceosome and can undergo further processing, such as nuclear export or degradation.
Regulation by RNA-Binding Proteins
In simple terms: Helper proteins can speed up or slow down the circle-making process.
RNA-binding proteins (RBPs) such as QKI, MBL, and HNRNPL regulate back-splicing by binding to intronic sequences and promoting or inhibiting splice site pairing. For example, QKI dimerization brings flanking introns together to enhance circRNA formation. Conversely, some RBPs can compete with spliceosome components and reduce circRNA production.
Coupling with Transcription and Linear Splicing
In simple terms: Circle-making happens at the same time as normal splicing and can compete with it.
Back-splicing is co-transcriptional and can compete with canonical linear splicing. The relative abundance of circRNA versus linear mRNA is determined by the balance between these two processes, which is influenced by transcription elongation rates and splicing factor availability. This coupling ensures that circRNA production is integrated with overall gene expression programs.
Key Genes Involved in GO:0160091 spliceosome-depend formation of circular RNA
The following genes and proteins are key players in the spliceosome-dependent formation of circular RNAs, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| QKI | RNA-binding protein that promotes back-splicing by dimerizing and bringing splice sites together | Knockout reduces circRNA levels; overexpression increases them |
| MBL | Muscleblind-like protein that regulates circRNA biogenesis in muscle and brain | Point mutations affect RNA binding and circRNA production |
| HNRNPL | Heterogeneous nuclear ribonucleoprotein L that binds intronic elements to enhance back-splicing | Knockdown alters circRNA profiles |
| SF3B1 | Core spliceosome component; mutations affect back-splicing efficiency | Knockout is lethal; point mutations linked to cancer |
| U2AF1 | Splicing factor that recognizes 3' splice sites and influences circRNA formation | Mutations associated with myelodysplastic syndromes |
| SRSF1 | Serine/arginine-rich splicing factor that modulates splice site selection | Overexpression changes circRNA/mRNA ratios |
| DHX9 | RNA helicase that resolves double-stranded RNA structures and inhibits circRNA formation | Knockdown increases circRNA levels |
| ADAR1 | Adenosine deaminase that edits dsRNA and suppresses circRNA biogenesis | Knockout leads to increased circRNA production |
| EIF4A3 | Exon junction complex component that regulates circRNA export and stability | Knockdown affects circRNA nuclear export |
| XPO1 | Nuclear export receptor that mediates circRNA export | Inhibition blocks circRNA cytoplasmic localization |
| NXF1 | Nuclear export factor that binds circRNAs for export | Knockdown reduces cytoplasmic circRNA levels |
| UPF1 | Nonsense-mediated decay factor that can degrade circRNAs | Knockdown increases circRNA stability |
| DROSHA | Microprocessor component that can cleave circRNAs | Knockout alters circRNA abundance |
| DGCR8 | Microprocessor component that binds circRNAs | Knockdown affects circRNA processing |
| PTBP1 | Polypyrimidine tract-binding protein that regulates alternative splicing and circRNA formation | Knockdown changes circRNA patterns |
| TDP-43 | RNA-binding protein linked to neurodegeneration; affects circRNA biogenesis | Mutations associated with ALS |
| FUS | RNA-binding protein involved in splicing and circRNA regulation | Mutations linked to ALS |
| CIRBP | Cold-inducible RNA-binding protein that modulates circRNA production | Overexpression increases specific circRNAs |
How Is spliceosome-depend formation of circular RNA Regulated?
The spliceosome-dependent formation of circular RNAs is regulated at multiple levels. Cis-acting elements such as complementary intronic sequences and RNA editing by ADAR1 can inhibit back-splicing. Trans-acting factors including RNA-binding proteins (e.g., QKI, MBL, HNRNPL) and spliceosome components modulate the efficiency of back-splicing. Transcription elongation rates and chromatin modifications also influence the competition between linear and back-splicing. Additionally, cellular stress conditions and signaling pathways can alter circRNA production, although specific pathways like mTOR or ISR have not been definitively linked to GO:0160091 in the provided literature.
spliceosome-depend formation of circular RNA and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SF3B1 | Myelodysplastic syndromes, leukemia | Knock-in of SF3B1 mutations in hematopoietic stem cells |
| U2AF1 | Myelodysplastic syndromes | Point mutation knock-in in cell lines |
| TDP-43 | Amyotrophic lateral sclerosis | Knockout or overexpression in neuronal cells |
| FUS | Amyotrophic lateral sclerosis | Knock-in of ALS-associated mutations |
| QKI | Cancer, cardiovascular disease | Knockout and overexpression in cancer cell lines |
Circular RNAs in Cancer
Dysregulation of circRNA biogenesis is frequently observed in cancer. For example, the oncogenic fusion protein EML4-ALK increases circRNA production, and specific circRNAs such as circHIPK3 promote cell proliferation. Mutations in spliceosome genes like SF3B1 and U2AF1, which are common in myelodysplastic syndromes and leukemias, can alter back-splicing patterns and contribute to disease pathogenesis. Targeting circRNA biogenesis pathways may offer therapeutic opportunities.
Circular RNAs in Neurodegeneration
CircRNAs are highly enriched in the brain, and their dysregulation has been implicated in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). RNA-binding proteins like TDP-43 and FUS, which are mutated in ALS, regulate circRNA formation, and their dysfunction leads to altered circRNA profiles. These findings suggest that circRNA biogenesis is critical for neuronal health.
Circular RNAs in Cardiovascular Disease
CircRNAs such as circANRIL and circFOXO3 are involved in atherosclerosis and cardiac hypertrophy. The spliceosome-dependent formation of these circRNAs is regulated by factors like QKI and MBL, which are expressed in muscle and heart tissues. Modulating circRNA levels may provide new strategies for treating cardiovascular disorders.
From spliceosome-depend formation of circular RNA-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does knockout of QKI reduce circRNA levels? | QKI knockout cell line via CRISPR |
| Does a point mutation in SF3B1 alter back-splicing? | SF3B1 point mutation knock-in |
| Can a tagged spliceosome component be used to pull down circRNAs? | Tagged knock-in of SF3B1 or U2AF1 |
| Does overexpression of MBL increase specific circRNAs? | MBL overexpression vector |
| What is the effect of ADAR1 knockout on circRNA biogenesis? | ADAR1 knockout cell line |
| Can CRISPR library screening identify novel circRNA regulators? | Genome-wide CRISPR knockout library |
How to Study the spliceosome-depend formation of circular RNA Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | CircRNA expression and back-splice junctions | Tissue-specific circRNA profiling |
| Ribo-seq | Translation of circRNAs | Identification of circRNA-encoded proteins |
| CLIP-seq | RNA-binding protein binding sites | Mapping QKI or MBL binding on pre-mRNAs |
| RNA FISH | Subcellular localization of circRNAs | Nuclear vs cytoplasmic distribution |
| Mass spectrometry | Proteins associated with circRNAs | Identifying circRNA-protein complexes |
| CRISPR knockout | Gene function in circRNA biogenesis | Testing candidate regulators |
| CRISPR knock-in | Tagged protein expression | Endogenous tagging of spliceosome components |
| Overexpression | Gain-of-function effects | Increasing circRNA levels for functional studies |
RNA Sequencing and circRNA Detection
RNA-seq followed by computational tools such as CIRI, find_circ, or CIRCexplorer is used to identify back-splice junctions and quantify circRNA expression. These methods rely on the unique BSJ reads that span the circularization site. Tissue-specific and developmental stage-specific circRNA profiles can be obtained.
Ribo-Seq and Translation Analysis
Ribo-seq can detect ribosome footprints on circRNAs, revealing their translation potential. This method has shown that some circRNAs are translated into proteins in a cap-independent manner. Combining Ribo-seq with circRNA-specific knockdown can validate translation events.
Proteomics and RNA-Protein Interaction
Mass spectrometry-based proteomics and RNA pull-down assays identify proteins that bind to circRNAs or regulate back-splicing. CLIP-seq and RIP-seq can map the binding sites of RNA-binding proteins on pre-mRNAs. These approaches reveal the regulatory network of circRNA biogenesis.
Imaging and Localization Studies
Single-molecule RNA FISH and live-cell imaging with fluorescently labeled circRNAs can visualize their subcellular localization and dynamics. Nuclear export of circRNAs can be tracked using reporter systems. These methods are useful for studying the spatial regulation of circRNA formation and function.
How CRISPR Can Be Used to Study GO:0160091 spliceosome-depend formation of circular RNA
Knockout
CRISPR knockout of genes involved in back-splicing, such as QKI or SF3B1, can reveal their essential roles in circRNA formation. However, knockout of core spliceosome genes may be lethal, requiring inducible systems. Knockout of ADAR1 or DHX9 increases circRNA levels, confirming their inhibitory roles.
Point Mutation
Point mutations in spliceosome genes (e.g., SF3B1 K700E) can be introduced using CRISPR base editing or HDR to study their impact on back-splicing. These models mimic cancer-associated mutations and help dissect the molecular mechanisms of altered circRNA biogenesis.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) into endogenous spliceosome genes allows for affinity purification of circRNA-protein complexes. Knock-in of reporter constructs with specific intronic elements can be used to study cis-regulation of back-splicing.
Overexpression
Overexpression of RNA-binding proteins like QKI or MBL using CRISPR activation (CRISPRa) or lentiviral vectors can enhance circRNA production. Overexpression of engineered circRNAs is also used for therapeutic applications.
How EDITGENE Supports spliceosome-depend formation of circular RNA Research
Researchers studying spliceosome-depend formation of circular RNA-related genes often need to determine whether a candidate gene is causally involved in circRNA biogenesis or whether its manipulation alters circRNA levels and function. EDITGENE provides a comprehensive suite of CRISPR-based services to enable such functional studies.
Contact EDITGENE today to design your custom CRISPR model for spliceosome-depend formation of circular RNA research.
Frequently Asked Questions About spliceosome-depend formation of circular RNA
What is GO:0160091?
GO:0160091 is a Gene Ontology term for the spliceosome-dependent formation of circular RNAs by back-splicing of pre-mRNAs.
What genes are involved in spliceosome-depend formation of circular RNA?
Key genes include QKI, MBL, HNRNPL, SF3B1, U2AF1, and ADAR1, which regulate back-splicing.
How are circular RNAs formed?
Circular RNAs are formed by back-splicing, where a downstream splice donor is joined to an upstream splice acceptor in a spliceosome-dependent manner.
What is the role of the spliceosome in circRNA formation?
The spliceosome catalyzes the transesterification reactions that ligate the splice sites to form the circular RNA.
Are circular RNAs translated?
Some circular RNAs can be translated into proteins via cap-independent mechanisms.
What diseases are associated with circRNA biogenesis?
Dysregulated circRNA formation is linked to cancer, neurodegeneration, and cardiovascular diseases.
How can I study circRNA formation using CRISPR?
CRISPR knockout, point mutation, knock-in, and overexpression models can be used to dissect gene function in circRNA biogenesis.
What methods detect circular RNAs?
RNA-seq with back-splice junction analysis, Ribo-seq, and RNA FISH are commonly used.
Is circRNA formation conserved?
Yes, back-splicing and circRNA production are conserved across eukaryotes.
Can circRNAs be used therapeutically?
Engineered circRNAs are being developed as stable therapeutic agents for various diseases.
Conclusion
GO:0160091, spliceosome-depend formation of circular RNA, represents a fundamental biological process that generates a diverse class of regulatory RNA molecules. Understanding the molecular mechanisms, key genes, and regulatory factors involved in back-splicing is essential for elucidating circRNA functions in health and disease. With the advent of CRISPR-based tools and advanced sequencing methods, researchers can now dissect this process with unprecedented precision. EDITGENE provides comprehensive services to support these investigations, from knockout and knock-in models to library screening and bioinformatics.
References
- 1. Hwang HJ et al.. 2024. Molecular mechanisms of circular RNA translation.. Exp Mol Med 56(6):1272-1280 PMID: 38871818
- 2. Szabo L et al.. 2015. Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.. Genome Biol 16(1):126 PMID: 26076956
- 3. Liu CX et al.. 2022. Circular RNAs: Characterization, cellular roles, and applications.. Cell 185(12):2016-2034 PMID: 35584701
- 4. Wesselhoeft RA et al.. 2018. Engineering circular RNA for potent and stable translation in eukaryotic cells.. Nat Commun 9(1):2629 PMID: 29980667
- 5. Ngo LH et al.. 2024. Nuclear export of circular RNA.. Nature 627(8002):212-220 PMID: 38355801
- 6. Obi P et al.. 2021. The design and synthesis of circular RNAs.. Methods 196:85-103 PMID: 33662562
- 7. O'Leary E et al.. 2025. The therapeutic potential of circular RNAs.. Nat Rev Genet 26(4):230-244 PMID: 39789148
- 8. Li X et al.. 2018. The Biogenesis, Functions, and Challenges of Circular RNAs.. Mol Cell 71(3):428-442 PMID: 30057200
