Circular RNAs (circRNAs) are regulatory molecules that show diverse functions. However, the regulation of circRNA formation is not yet well-understood. Through large-scale neuron isolation from the first larval stage of Caenorhabditis elegans followed by RNA sequencing
with ribosomal RNA depletion, the first neuronal circRNA profile in C. elegans was obtained. Using circRNAs identified in this dataset, I performed an in vivo investigation of circRNA regulation by cis and trans elements. Several neuronal circRNAs were knocked out by deleting one of the reverse complementary match (RCM) sequences flanking circRNA exon(s) (cis elements). Further, RCMs not only vigorously promote circRNA formation but also are beneficial for the skipping of exon(s) to be circularized. 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 exonskipping is not absolutely required for back-splicing, neither the other way. Instead, the coupled exon-skipping and back-splicing are promoted by RCMs directly at the same time. As for trans elements that regulate circRNA in C. elegans, thirteen RNA binding proteins were screened, among which loss of FUST-1, the homolog of FUS, causes substantial downregulation of multiple circRNAs. Further, FUST-1 regulates circRNAs without affecting their cognate linear mRNA levels. When recognizing circRNA pre-mRNAs,
FUST-1 can affect the coupled exon-skipping and circRNA formation in the same genes. In zip-2, the 5’ splice sites for back splicing and exon skipping seem important for FUST-1’s role in exon-skipping and back-splicing regulation, respectively. Two mutations (R446S and
P447L) were introduced in FUST-1 to mimic the amyotrophic lateral sclerosis-related natural mutations in the nuclear localization signal of FUS (R524S and P525L). Both mutations dramatically affect circRNA levels. Moreover, I identified an autoregulation loop
important for circRNA regulation in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different Nterminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation. This thesis explored circRNA regulation in vivo using C. elegans as the model organism, providing new insights into mechanisms governing the relationship between back-splicing. The combinatorial regulation of circRNA by cis and trans elements supports a model of circRNA formation, where RCM sequences (cis elements) determine whether circRNA can be formed or not, and RBPs (trans elements) regulate how efficiently they can be produced.
Exam Date
2021-08-05
Degree Conferral Date
2021-09-30
Degree
Doctor of Philosophy
Degree Referral Number
38005甲第80号
Degree Conferrral Institution
Okinawa Institute of Science and Technology Graduate University
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Investigation of Circular RNA Regulation by Cis and Trans Elements in Caenorhabditis Elegans
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