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Research Article
Dong Cao
Okinawa Institute of Science and Technology Graduate University
Corresponding Author
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Background: Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Although circRNAs were found in C. elegans, the neuron-specific circRNA data is not available yet. Exon-skipping is found to be correlated to circRNA formation, but the mechanisms that link them together are not clear.
Results: Here, through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the first neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of RCMs in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that back-splicing still happened without exon-skipping, and vice versa.
Conclusions: For the first time, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are likely derived from neuronal genes. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.
Keywords
circular RNA, back-splicing, exon-skipping, reverse complementary matches, FACS
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No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.docx
Additional file 1: Complete supplemental figures. Figure S1. Dissociation and sorting of L1 worms. Figure S2. circRNA analysis and experimental validation. Figure S3. circRNA-flanking intron analysis and RCM deletions in circRNA genes. Figure S4. Skipped transcripts in circRNA genes. Figure S5. Effect of RCM-deletion on exon-skipping. Figure S6. Gene structures of zip-2 ortholog genes. Figure S7. Sequence confirmation of mutated ss and BP sites in zip-2. Figure S8. Orignial full images of northern blot and agarose gels.
- Additionalfile2.xlsx
Additional file 2: Table S1. Strains used in this study.
- Additionalfile3.xlsx
Additional file 3: Table S2. Primers used in this study.
- Additionalfile4.xlsx
Additional file 4: Table S3. List of gRNA sequences, recombinant oligos and validation primers used for mutagenesis by CRISPR-Cas9.
- Additionalfile5.xlsx
Additional file 5: Table S4. Differential expression analysis results of linear mRNAs between the sort and the whole group by DESeq2.
- Additionalfile6.xlsx
Additional file 6: Table S5. List of filtered circRNAs with BSJ reads in each sample.
- Additionalfile7.xlsx
Additional file 7: Table S6. Differential expression analysis results of filtered circRNAs between the sort and the whole group by DESeq2.
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A new preprint design is coming!
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See the published version of this preprint at BMC Genomics.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Dong Cao
Okinawa Institute of Science and Technology Graduate University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Genomics.
Version 1
Posted 12 May, 2021
No community comments so far
Editorial decision: Major revision
On 02 Jun, 2021
Reviews received
Received 02 May, 2021
Reviewers agreed
On 14 Apr, 2021
Reviews received
Received 11 Apr, 2021
Reviewers agreed
On 09 Apr, 2021
Reviewers invited
Invitations sent on 09 Apr, 2021
Editor assigned
On 07 Apr, 2021
Editor invited
On 07 Apr, 2021
Submission checks complete
On 06 Apr, 2021
First submitted
On 24 Mar, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Epigenetics & Genomics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Genomics.
Background: Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Although circRNAs were found in C. elegans, the neuron-specific circRNA data is not available yet. Exon-skipping is found to be correlated to circRNA formation, but the mechanisms that link them together are not clear.
Results: Here, through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the first neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of RCMs in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that back-splicing still happened without exon-skipping, and vice versa.
Conclusions: For the first time, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are likely derived from neuronal genes. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
No competing interests reported.
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.docx
Additional file 1: Complete supplemental figures. Figure S1. Dissociation and sorting of L1 worms. Figure S2. circRNA analysis and experimental validation. Figure S3. circRNA-flanking intron analysis and RCM deletions in circRNA genes. Figure S4. Skipped transcripts in circRNA genes. Figure S5. Effect of RCM-deletion on exon-skipping. Figure S6. Gene structures of zip-2 ortholog genes. Figure S7. Sequence confirmation of mutated ss and BP sites in zip-2. Figure S8. Orignial full images of northern blot and agarose gels.
- Additionalfile2.xlsx
Additional file 2: Table S1. Strains used in this study.
- Additionalfile3.xlsx
Additional file 3: Table S2. Primers used in this study.
- Additionalfile4.xlsx
Additional file 4: Table S3. List of gRNA sequences, recombinant oligos and validation primers used for mutagenesis by CRISPR-Cas9.
- Additionalfile5.xlsx
Additional file 5: Table S4. Differential expression analysis results of linear mRNAs between the sort and the whole group by DESeq2.
- Additionalfile6.xlsx
Additional file 6: Table S5. List of filtered circRNAs with BSJ reads in each sample.
- Additionalfile7.xlsx
Additional file 7: Table S6. Differential expression analysis results of filtered circRNAs between the sort and the whole group by DESeq2.
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