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. 2019 May 21;47(9):4586-4596.
doi: 10.1093/nar/gkz155.

Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters

Teressa Paulsen  1 , Yoshiyuki Shibata  1 , Pankaj Kumar  1 , Laura Dillon  1 , Anindya Dutta  1
Affiliations

Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters

Teressa Paulsen et al. Nucleic Acids Res. .

Abstract

Interest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) in mammalian tissues and cell lines are too small to carry protein coding genes. We have tested functional capabilities of microDNA by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA are transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA that carry miRNA genes form transcripts that are processed by the endogenous RNA-interference pathway into mature miRNA molecules, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA that contain sequences of exons repress the endogenous gene from which the microDNA were derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits, POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.

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Figures

Figure 1.
Figure 1.
(A) Diagram of artificial microDNA creation by LAMA. (B) Circular and linear products of LAMA run on a denaturing PAGE gel before and after ligation cycles. Circular DNA accumulates to form ssDNA, nicked and supercoiled dsDNA. (C) 32P-UTP-labeled RNA run on PAGE gel after in vitro transcription assay. Products are seen at size ranges of multiples of the microDNA length. Ss: single-stranded; ds: double-stranded; sc: supercoiled; rel: relaxed or nicked; circ: circular; NE: HeLa nuclear extract. Representative replicate of duplicates.
Figure 2.
Figure 2.
(A) Diagram of transcription of microDNA carrying only the promoterless pre-microRNA part of the microRNA gene. The transcripts are processed by endogenous RNA interference proteins into functional mature miRNA (B) Expression of pre-microRNA molecules after the addition of indicated amounts of the corresponding artificial microDNA molecules. Expression is relative to β-actin (hsa-mir-191 and hsa-mir-126) and GAPDH (hsa-mir-145) and normalized again to the negative control of a transfected GFP plasmid. Mean and S.E. of three transfections. (C) Expression of processed mature miRNA molecules after transfection of indicated amounts of artificial microDNA molecules. Expression is relative to β-actin (hsa-mir-191 and hsa-mir-126) and GAPDH (hsa-mir-145) normalized to a negative control of a transfected GFP plasmid. Mean and S.E. of three transfections. (D) Expression of the + and the – strand of the pre-microRNA, hsa-mir-145, from microDNA molecules relative to GAPDH. Strand-specific primers were used for the reverse-transcription to form cDNA specifically from the (+) or (-) strand of RNA. Results from three transfections.
Figure 3.
Figure 3.
(A) Transfection of artificial microDNA, carrying indicated pre-miRNA sequences, to 293T cells decreases expression of a co-transfected Renilla luciferase reporter containing a sequence complementary to the miRNA sequence within its 3′ UTR. RL activity expressed relative to a co-transfected firefly luciferase and normalized again to the level in cells transfected with 0 ng of microDNA. Mean and S.E. of three experiments. * indicated P < 0.05 in a Student’s t-test. (B) Repression of luciferase in dual-luciferase assay is observed in WT 293T cells but not in DICER1-/- 293T cells. Mean and S.E. of three experiments. * indicated P < 0.05 in a Student’s t-test. (C) Endogenous cellular targets of indicated miRNAs are repressed after transfection of the synthetic microDNA carrying the indicated pre-miRNA genes. mRNAs quantitated by QRT-PCR and expressed relative to the β-actin gene and normalized to the level in cells transfected with 0 ng microDNA. Mean and S.E. of three experiments.
Figure 4.
Figure 4.
(A) Diagram of the theoretical mechanism of the formation of si-like or sh-like RNA from microDNA containing an exonic sequence either from transcription of both strands of the microDNA or from folding of the transcript into a short-hairpin. (B) Transfection of microDNA containing the sequence of exon 2 of the TESC-AS1 gene represses the expression of the TESC-AS1 gene in 293Tcells. The repression is observed in 293T cells but not in DICER-/- 293T cells. The same is seen with microDNA carrying a portion of (C) Exon 1 of KCNQ1OT1 and (D) the Exon 6 of SIGLEC9. The RNAs were quantitated by QRT-PCR, and the values expressed relative to β-actin and normalized to the cells with 0 ng of transfected microDNA. Mean and S.E. of three experiments. *P < 0.05 in a Student’s t-test.
Figure 5.
Figure 5.
Efficiency of pull-down of RNA polymerase complex by HT. Detection of RNA polymerase III catalytic subunit (POLR3A) in the HT-POLR2H or HT-POLR3F pull-down. HaloTag-fused RNA polymerase subunit POLR2H or POLR3F was expressed in 293A cells (asterisk). The HT-tagged polymerase subunits were covalently bound to the HaloLink resin, and the non-covalently associated proteins were eluted by boiling in Laemmli Sample buffer. The HT-proteins are covalently bound to the beads and are mostly not eluted. Some covalent bonds break to release traces of the HT protein in the eluate (squares). However, non-covalently associated POLR3A of the RNA polymerase complex is specifically released in the eluates from the HT-POLR3F and HT-POLR2H pull-downs.
Figure 6.
Figure 6.
Subunits of PolI, PolII and PolIII bind to microDNA. (A) Diagram of pull-down of Halo-tagged RNA polymerase subunits and purification of associated microDNA for rolling circle amplification with random hexamers (RCA). (B) RCA products were sheared, ligated to library primers for high-throughput sequencing, amplified by PCR for the indicated cycles and comparable aliquots run on a gel and visualized by ethidium bromide fluorescence. (C) Complexity of microDNA in the libraries prepared from the POLR3F and POLR2H pull-downs relative to the tag-only control as measured in Table 1. The error bars indicate the S.D. from 10 random subsamples from each library. (D–F) Characterization of microDNA molecules pulled down by POLR3F and POLR2H. (D) Enrichment relative to random expectation of the microDNA from areas of the genome with indicated genomic features, (E) length distribution and (F) GC content.
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