Circular RNA Translation
I published it here. There has always been some recent news circulating on social media regarding different RNA biotypes. I am lucky to meet incredible researchers from my current lab at UCC to discuss something exciting every time. Moreover, I have been reading about circular RNAs (circRNA) for a while. I would like to share my understanding of the “translation of circular RNA”.
Fasten your belt because we need to learn some basics before diving in! There are several aspects. I will not go into the details of circRNA biogenesis.
Before describing the typical/well-known/canonical or less-known/non-canonical modes of translation, let’s understand why translation matters first. If you are familiar with the central dogma, DNA*->(transcription)RNA->(translation)protein, proteins are the critical functional units/doers of a cell.
Fun fact: Although there are various short and long RNA biotypes, we usually refer to mRNA when we talk about coding, and the rest are known as non-coding biotypes. The part of mRNA where it is translated into protein is called coding sequences/region (CDS).
Let's go back to the existential question of this post!
Does circular RNA even translate?
If yes, how do we know?
Modes of Translation in Eukaryotes
The best-known cap-independent translation initiation mechanisms are promoted by Internal Ribosome Entry Sites (also known as IRES) through
either IRES binding auxiliary trans-acting sites (ITAFs) or epigenetic marks such as N6-methyladenosine (m6A) modification (which then interacts with/recruits some other translation initiation factors) at potential upstream open reading frames (uORF) within 5' Untranslated Region (UTR).
There are typical and less-known ways of how to read (or translate) the information shared in RNA: cap-dependent and cap-independent translation (CIT).
There are two crucial components of mRNA for the stability and processing during cap-dependent translation of mRNA:
- 5'methyl guanosine (m7G) cap
- poly-A tail
However, it turns out that there are alternative mechanisms present for RNA translation, especially when the canonical translation is somehow blocked (i.e., cap initiation factors are not able to be recruited or shut down due to viral infection) or less efficient. These can be categorized as non-canonical ways of translation. There are still lots of unknowns about them.
The best-known cap-independent translation initiation mechanisms are promoted by Internal Ribosome Entry Sites (also known as IRES) through
either IRES binding auxiliary trans-acting sites (ITAFs) or epigenetic marks such as N6-methyladenosine (m6A) modification (which then interacts with/recruits some other translation initiation factors) at potential upstream open reading frames (uORF) within 5' Untranslated Region (UTR).
Most of the translation relies on cap-dependent translation. The cap-independent translation usually takes part in certain abnormal conditions such as starvation, oxidative stress, and heat shock.
This is where circRNAs become interesting.
Fun fact: As endogenous IRES might be rare, some researchers proposed short IRES-like elements sufficient to drive the cap-independent translation.
Why Does circRNA Matter
Yet, the recent advancements in sequencing, data analysis, experimental techniques such as ribosome profiling, and studies on the potential of cap-independent translation showed that some circular RNAs are coding proteins and translated.
As the name suggests, some RNA has a single-stranded unique circular structure and is expressed endogenously, naturally found in cells. Since its 5' and 3' ends are covalently linked as a loop and lack 5' or 3' free ends, circRNA has shown to have better stability compared to mRNA counterparts (e.g., against exonucleases). However, circRNA was proposed as a non-coding RNA biotype with no known protein-coding function until the last decade.
Yet, the recent advancements in sequencing, data analysis, experimental techniques such as ribosome profiling, and studies on the potential of cap-independent translation showed that some circular RNAs are coding proteins and translated.
As circular closed-loop RNA must rely on cap-independent translation (CIT), it will able to be easily detected/used in conditions stimulating CIT.
Fun fact: RNA can also be circularized synthetically as a therapeutic tool with enzymatic ligation or self-splicing.
How Do We Know circRNA Translates?
There are several good practices have been developed over the years regarding how to predict or prove the translation of natural cirRNAs:
- ribosome/polysome binding/occupancy
- experimentally mapped translation initiation sites (TIS)
- (potential) internal ribosome entry site (IRES)
- (potential) N-6-methyladenosine modification sites (m6A) to promote translation initiation
- (potential) open reading frames (ORF)
- mass spectrometry (MS) detecting the circRNA encoded peptides across back-splice junctions (BSJ)
Fun fact: You might be wondering how people can make sure whether they detect any coding linear RNA or circRNA. First, exonuclease treatment and several other approaches are used to digest linear RNA/enrich circRNA during the isolation process. Secondly, most endogenous circRNA are back-spliced(BS) from linear counterparts, so total-RNA seq data with detecting BS events or long-read sequencing is useful.
The last time I took an up-to-date molecular biology course was ~10 years ago when textbook information was not updated on certain topics. I think the field is advancing quite fast. I enjoyed refreshing my knowledge of the basics and application of our current understanding of RNA therapeutics. I hope you have fun reading it, too!
References/Further Reading:
- Why does translatable circular RNA matter? :
The therapeutic potential of circular RNAs, https://www.nature.com/articles/s41576-024-00806-x
- Central dogma, nucleic acid, protein, translation (briefly): https://www.genome.gov/genetics-glossary/Central-Dogma#:~:text=The%20central%20dogma%20of%20molecular,National%20Human%20Genome%20Research%20Institute
- My previous post about the non-canonical way of translation summarized an article: https://www.linkedin.com/posts/fbdincaslan_the-dark-proteome-translation-from-noncanonical-activity-7271979603877871616-KGiU?
- A nice YouTube tutorial video about Cap-Independent Translation Initiation — IRES (virus and eukaryote) and uORF, https://www.youtube.com/watch?v=3nhOGTLpEi4
- If you ever wonder why you don’t capture circular RNA translation in regular conditions that much (a few examples with relevant discussions):
CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs, https://www.nature.com/articles/srep31313
Hypoxia-induced circRNF13 promotes the progression and glycolysis of pancreatic cancer, https://www.nature.com/articles/s12276-022-00877-y
A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis, https://www.nature.com/articles/s41388-017-0019-9#Sec2
- Translation of CircRNAs, https://www.sciencedirect.com/science/article/pii/S1097276517301363?via%3Dihub
- Pervasive translation of circular RNAs driven by short IRES-like elements, https://www.nature.com/articles/s41467-022-31327-y
- Where RNA modifications, specifically the methylation, join the chat:
Extensive translation of circular RNAs driven by N6-methyladenosine, https://www.nature.com/articles/cr201731
Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis, https://www.sciencedirect.com/science/article/pii/S1097276517301326?via%3Dihub
- A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma, https://www.nature.com/articles/s41467-018-06862-2
- Structured elements drive extensive circular RNA translation, https://www.sciencedirect.com/science/article/pii/S1097276521006262
- Fantastic circRNAs and Where to Find Them: Some circRNA identification tools
CircPrimer 2.0: a software for annotating circRNAs and predicting translation potential of circRNAs, https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-022-04705-y
Full-length circular RNA profiling by nanopore sequencing with CIRI-long, https://www.nature.com/articles/s41596-023-00815-w
Best practice standards for circular RNA research, https://www.nature.com/articles/s41592-022-01487-2#Tab2
An updated resource for the detection of protein-coding circRNA with CircProPlus, https://www.nature.com/articles/s41598-024-69744-2
circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing, https://elifesciences.org/articles/69457
TransCirc: an interactive database for translatable circular RNAs based on multi-omics evidence, https://academic.oup.com/nar/article/49/D1/D236/5930392?login=true (I liked the database idea so much. However, they lack links to refer to evidence where claimed, which article, etc.)
- The molecular basis of translation initiation and its regulation in eukaryotes, https://www.nature.com/articles/s41580-023-00624-9
