Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Summary

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Abstract

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Introduction

Circular RNAs
Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are expressed in most organisms. They are generated by joining a downstream 5' splice site to an upstream 3' splice site, a process called back-splicing (Figure 1A)¹. Sequences in the pre-mRNA that exhibit base complementary as short as 30-40 nt bring back-splice sites into proper alignment for circRNA formation². In humans, Alu elements¹, representing about 11% of the genome³, form extensive double str....

Protocol

1. Design of the constructs

1. Use the UCSC genome browser²⁴ to identify repetitive elements necessary for circular RNA formation and incorporate them in the constructs. Importantly, primers for amplification need to be outside the repetitive elements.
2. Paste the circular RNA sequence (Supplemental Figure 1 is a test sequence) into https://genome.ucsc.edu/cgi-bin/hgBlat?command=start and select the right organism. Submit the sequence and go to browser view, zoom out 1.5x or as appropriate (Figure 2<....

Results

Reporter genes allow determination of regulatory factors that influence circular RNA formation. However, these reporter genes are large and contain repetitive elements that often make DNA constructs unstable. Due to their large size, it is often necessary to delete parts of the introns, which is achieved by amplifying genomic pieces containing the exons and smaller flanking intronic parts. These DNA pieces are enzymatically assembled, allowing construction without restriction enzymes.

The exam.......

Discussion

In general, circular RNAs are low abundant¹, which complicates the study of their function and formation. Similar to linear RNAs¹³, the use of reporter minigenes allows the identification of cis and trans-acting factors that regulate the formation of circular RNAs. Thus, this approach generates hypotheses that can be further tested using the endogenous genes.

The most critical step is the design of the reporter gene. The enzymatic assembly of DNA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(PEI) Hydrochloride	Polysciences	24765-1
Builder tool	NEB		https://nebuilder.neb.com/#!/
Dark Reader Transilluminator.	Clare Chemical Research
Enzymatic DNA assembly kit	NEB	E2621S
Gel and PCR cleanup kit	Promega	A9282
Glyco Blue	Thermo Fisher	AM9516
pcDNA3.1 cloning site	Polycloning site		https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf
Polymerase 1	NEB	M0491L	Q5 DNA polymerase
Polymerase 2	Biorad	1725310	Long range polymerase (NEB), iproof (BioRad)
Polymerase 2	Qiagen	206402	Qiagen long range polymerase kit
Reverse Transcriptase	Thermo Fisher	18080044
RNA isolation kit	Life Technologies	12183025	Ambion by Life Technologies
RNAse R	Lucigen	RNR07250	Epicenter/Lucigen
Stable competent cells	NEB	C3040H	NEB stable cells
Standard cloning bacteria	NEB	C2988J	NEB5-alpha competent
Web tool to design primers	NEB		https://nebuilder.neb.com/#!/
Web-based temperature calculations	NEB		https://tmcalculator.neb.com/#!/main