Ruibo Bio: Introduction to the method and function of circular RNA research

Rui Bo creature: a ring RNA- RNA secret unknown parallel universe

Cyclic RNA, known as the hidden unknown RNA parallel universe. Recently, Dr. Zheng Qiupeng from Fudan University (one of the guests of the 2016 exchange meeting) released their latest research on circHIPK3 at Nature Communications as the first author. Now, let's learn the research ideas of circRNA with Xiaobian and understand the RNA world reshaped by the circle.

Sample selection and analysis methods for circular RNA sequencing

RNA sequencing analysis from 6 normal tissues (brain, colon, heart, liver, lung, stomach) and 7 cancer tissues (bladder urothelial carcinoma, breast cancer, colorectal cancer, liver cancer, gastric cancer, renal clear cell carcinoma, prostate cancer) Of total RNA after deribosomal RNA, 67,358 potential circular RNAs were found, of which 27,296 contained at least two unique back-spliced ​​reads.

The researchers used RefSeq database 24 to annotate these circular RNAs; differences in circular RNA expression were calculated using the Wilcoxon rank-sum test to compare cancer samples with corresponding normal tissue samples.

Preliminary identification of predicted new circular RNA

To rule out that they are linear trans-splicing products, the researchers examined their physical properties. Outward-facing primers were designed for 36 co-expressed circular RNA transcripts to be identified. Each pair of primers was individually amplified with a different product from the HEK-293T cDNA. After RNase R treatment, all 36 back-spliced ​​sequences were apparent.

Which circular RNA is selected to continue downstream functional studies?

Abundance quantification is performed for each circular RNA in the sequencing data according to their linear form by Anchor alignment. The researchers examined their 5' and 3' end circular ratios (CR), with 5' CR>0.2; 3' CR>0.2; SRPBM>1 as the standard, 990 high abundance expressions were obtained. Circular RNA. The researchers noted that one of the circular RNAs from the HIPK3 gene Exon2, named circHIPK3, has a high abundance and back-spliced ​​ratio.

Multi-scale scale screening for high-abundance circRNA. Red and black dots indicate high abundance and low abundance circRNA, respectively, and circHIPK3 is shown with blue dots. The gray part is the screened high abundance circRNA.

The first step of circular RNA research: identification of ring

The researchers used outward-facing primers to amplify different products of the expected size and sequenced them by sanger sequencing. The expression level of the loop was detected by qRT-PCR, and the results were consistent with RNA-seq, and the expression levels in various tissues were higher than their linear forms.

The investigator then tested its stability and localization in HeLa cells. After treatment with Actinomycin D (transcriptional inhibitor), the circular RNA remained highly stable and resistant to RNase R exonuclease digestion, indicating that it It is indeed a ring form.

e) qRT-PCR showed circHIPK3 and HIPK3 mRNA in HeLa cytoplasm or nucleus. f) RNA FISH experiment of circHIPK3.

How is circHIPK3 formed?

Exon analysis on both sides of HIPK3 Exon2 revealed that the highly complementary Alu repeats were in the introns upstream of HIPK3 Exon2 with 28 short repeats (SINE) and 51 SINEs were downstream of Exon3. The researchers used CRISPR/Cas9 technology to remove the circHIPK3 flanking Alu sequence in HEK-293T cells. The results showed that the cyclization was inhibited after the downstream Alu sequence was deleted, but the upstream Alu deletion not only did not reduce the formation of circHIPK3, but also increased slightly. .

The schematic shows that the HIPK Exon2 region on the genome contains flanking Alu repeats and long introns. Alu elements and long introns were deleted using the CRISPR/Cas9 system (gRNA1-6). Primers on both sides of the gRNA are used to detect deletion effects (P1-5)

There are many Alu elements upstream of the primer loop exon. The researchers speculated that other Alu elements in the intron may promote cyclization, thus deleting the upstream large intron (gRNA5, gRNA6), resulting in a significant down-regulation of circHIPK3. There was no change in HIPK3 mRNA, demonstrating that deletion only affects back spilcing without affecting typical splicing, indicating that the flanking long introns with Alu complementary repeats are required for circHIPK3 production.

The sequence was deleted (gRNA3-6) using 4 pairs of gRNA, and normal PCR and qRT-PCR were performed. The primer site was designed outside the deletion region (P4+P5).

Ruibo Xiaobian said

How does RNA become circular?

a. During the variable shearing process, the exons migrate, shear to form the lasso structure, pull the shear site closer, and promote the sequence into loops.

b. Relying on adjacent reverse complementary sequence pairings, pulling the cleavage sites to attack each other and facilitating sequence looping

c. A single intron is directly looped

d. RNA-binding protein and trans-acting factors participate in loop formation

Research function, knock down to see

According to the literature, circular RNA can be knocked down by small interfering RNA. As shown in the figure below, the researchers used three siRNAs: a targeted backsplice sequence (si-circHIPK3), a targeted linear transcript (si-HIPK3), a ring that targets both linear and circular The exons (si-both) are provided by Ruibo . Next, the researchers observed the knockdown after cell proliferation assay and EdU staining (provided by Ruibo Bio) and found that si-circHIPK3 can inhibit several different cell growth, while si-HIPK3 cannot.

The DNA synthesis of Huh-7 cells was detected by the EdU assay after 48 hours of transfection of the three siRNAs.

circHIPK3 acts as a miRNA sponge

The AGO2 CLIP-Seq public data from doRiNA shows that AGO2 is located in the circHIPK3 region. The researchers performed pull down and luciferase reporter gene experiments, demonstrating that circHIPK3 may be an intermediate mediator of AGO2 and miRNA binding.

To find miRNAs that bind to circHIPK3, the researchers used luciferase to screen miRNA libraries. Of the 424 miRNAs, 9 miRNAs significantly attenuated luciferase reporter gene activity. Using TargetScan and PicTarmiRNA prediction software, they were found to contain a circHIPK3 region binding site. The researchers then mutated these binding sites and transfected mutant miRNAs did not reduce reporter gene activity. This result indicates that circHIPK3 may be a sponge of these miRNAs.

HEK-293T cells were transfected with 424 miRNA mimics and screened for luciferase reporter assays to identify whether they bind to circHIPK3. The nine miRNAs labeled in the figure are more effective in inhibiting enzyme activity.

It is worth noting that these nine miRNAs can inhibit cell growth, and miR-124 is the most effective. Therefore, the researchers used biotin-conjugated miR-124 mimic (provided by Ruibo Bio) to enrich a large number of circHIPK3. The results of several experiments show that circHIPK3 can directly bind to miR-124 and inhibit its activity.

Left panel: HFH-124T cells transfected with 3'-streptavidin-conjugated miR-124, circHIPK3 levels in captured cell lysates; right panel: transfected si-cHIPK3, miR-124 or cHIPK3 vectors HEK-293T cells were assayed for expression of IL6R and DLX2 by qRT-PCR. IL6R and DLX2 are two known proliferation-promoting targets of miR-124, which are down-regulated with knockdown of circHIPK3, suggesting that circHIPK3 can rescue miR-124 from their inhibition.

Ruibo Xiaobian said

What is the function and role of circular RNA?

1. Regulate parental gene expression

Only intron ciRNA binds to pol II to promote gene transcription

ElciRNA with intron + exon forms a complex with U1 snRNP and binds to pol II to promote gene transcription

2. Act as ceRNA

Only exons of circRNA have MRE, which adsorb miRNA and regulate miRNA expression (miRNA sponge)

3. Biomarker as a disease

Shanghai Researcher: Exosomes also have circular RNA

4. Become a new disease treatment target

Original: Zheng Q, et al . Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun. 2016 Apr 6;7:11215.

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