Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer
Summary
The two different 3' rapid amplification of cDNA ends (3' RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3' UTR of a transcript using RNA obtained from different cancer cell lines.
Abstract
Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3' UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3' RACE. Expanded 3' RACE can detect unusual 3' UTRs, including gene fusions within the 3' UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.
Introduction
The formation of the 3' end is a critical step in mRNA maturation that comprises the cleavage of the pre-mRNA downstream of a PAS followed by the addition of ~250 untemplated adenines, which make up the poly(A) tail1,2. The poly(A) binding protein (PABP) binds to the poly(A) tail, and this protects the mRNA transcript from degradation, and facilitates translation1.
Current estimates suggest that 70% of human genes have multiple PASs, and thus undergo alternative polyadenylation, resulting in multiple 3' ends3. Thus, it is important to identify where the poly(A) tail attaches to the rest of the 3' UTR, as well as identify the PAS used by any given transcript. The advent of next-generation sequencing has resulted in the simultaneous identification of the 3' UTRs and the PASs of thousands of genes. This increase in sequencing capability has required the development of bioinformatic algorithms to analyze data involving alternative polyadenylation of the 3' end. For the de novo detection or validation of the PAS and hence mapping of the 3' end of individual genes from large scale sequencing data, 3' RACE remains the method of choice4,5. The sequences included in cDNA products of 3' RACE normally include only a portion of the 3' UTR that contains the poly(A) tail, the cleavage site, the PAS, and the sequences upstream of the PAS. Unlike PCR, which requires the design and use of gene specific forward and reverse primers, 3' RACE only requires two gene specific nested forward primers. Hence, PCR requires a more detailed knowledge of the nucleotide sequence of a large region of the gene being amplified4,6. Since 3' RACE uses the same reverse primer that targets the poly(A) tail for all polyadenylated RNA transcripts, only the forward primers need to be gene specific, thus, only requiring knowledge of a significantly smaller region of the mRNA. This enables the amplification of regions whose sequences are not fully characterized4,7. This has allowed 3' RACE to be used not only to determine the 3' end of a gene, but to also determine and characterize large regions upstream of the PAS that form a significant portion of the 3' UTR. By combining 5' RACE with the modified 3' RACE that includes larger portions of the 3' UTR and flanking regions, it is possible to fully sequence or clone an entire mRNA transcript from the 5' end to its 3' end8.
An example of this application of modified 3' RACE is the recent identification of a novel CCND1-MRCK fusion gene transcript from Mantle Cell Lymphoma cell lines and cancer patients. The 3' UTR consisted of sequences from both the CCND1 and MRCK genes and was recalcitrant to miRNA regulation9. The two nested CCND1 specific forward primers were complementary to the region immediately adjacent and downstream of the CCND1 stop codon. Although whole transcriptome sequencing together with specific bioinformatic tools can be used to detect gene fusions within the 3' UTR10, many labs may lack the financial resources or bioinformatic expertise to make use of this technology. Hence, 3' RACE is an alternative for de novo identification and validation of novel fusion genes involving the 3' UTR. Considering the drastic increase in the number of reported fusion genes as well as read through transcripts, 3' RACE has become a powerful tool in characterizing gene sequences11,12. In addition, recent studies have shown that different sequences within the 3' UTR as well as the length of the 3' UTR can affect mRNA transcript stability, localization, translatability, and function13. Due in part to an increased interest in mapping the transcriptome, there has been an increase in the number of different DNA polymerases being developed for use in the lab. It is important to determine what types of modifications can be made to the 3' RACE protocol in order to utilize the available repertoire of DNA polymerases.
This work reports adapting 3' RACE to map the entire 3' UTR, the PAS, and the 3' end cleavage site of the ANKHD1 transcript by using nested primers within the ANKHD1 section of the transcript and two different DNA polymerases.
Protocol
Representative Results
Discussion
Despite the advent of massive parallel sequencing technologies, on a gene-by-gene basis, 3' RACE still remains the easiest and most economical method to identify the PAS and nucleotides adjacent to the poly(A) tail. The adaptation described here expands using 3' RACE to both amplify and map sequences that include a portion of the ORF, the stop codon, and the entire 3' UTR of the ANKHD1 mRNA transcript. A major advantage of 3' RACE is that with a few minor adaptations, products from 3' RACE ca…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge Bettine Gibbs for her technical help.
Materials
| HeLa cells | ATCC | CCL-2 | Cervical cancer cell line. |
| Jeko-1 cells | ATCC | CRL-3006 | Mantle cell lymphoma cell line. |
| Granta-519 cells | DSMZ | ACC-342 | Mantle cell lymphoma cell line. |
| Fetal Bovine Serum | Sigma Aldrich | F6178 | Fetal bovine serum for cell culture. |
| Penicillin/Streptomycin | ThermoFisherScientific | 15140122 | Antibiotic and antimycotic. |
| GlycoBlue | Ambion | AM9545 | Coprecipitant. |
| DMEM | ThermoFisherScientific | 10569044 | Gibco brand cell culture media with GlutaMAX. |
| Nuclease Free-Water | ThermoFisherScientific | AM9938 | Ambion DNase and RNAse free water(non DEPC treated). |
| Dulbecco’s Phosphate-Buffered Solution | Corning | 21-030 | 1X PBS. |
| Chloroform | Sigma Aldrich | C7559-5VL | |
| 2-propanol | Sigma Aldrich | I9516 | |
| Reagent Alcohol | Sigma Aldrich | 793175 | Ethanol |
| Ethidium Bromide solution | Sigma Aldrich | E1510 | |
| TRIzol Reagent | ThermoFisherScientific | 15596026 | Monophasic phenol and guanidine isothiocyanate reagent. |
| 2X Extender PCR-to-Gel Master Mix | Amresco | N867 | 2X PCR-to-Gel Master Mix containing loading dye used in routine quick PCR assays and primer search. |
| 10mM dNTP | Amresco | N557 | |
| RQ1 RNase-Free DNase | Promega | M6101 | Dnase treatment kit. |
| Gel Loading Dye Orange (6X) | New England BioLabs | B7022S | |
| 2X Phusion High-Fidelity PCR Master Mix with HF buffer | ThermoFisherScientific | F531S | 2X PCR MasterMix containing a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase and other reagents. |
| PfuUltra II Fusion HS DNA polymerase | Agilent Technologies | 600670 | Modified DNA Polymerase from Pyrococcus furiosus (Pfu). |
| RevertAid RT Reverse Transcription Kit | Thermo Fischer | K1691 | Used for reverse transcription of mRNA into cDNA synthesis. Kit includes Ribolock RNAse inhibitor, RevertAid M-MuLV reverse transcriptase and other reagents listed in manuscript. |
| Pefect size 1Kb ladder | 5 Prime | 2500360 | Molecular weight DNA ladder. |
| Alpha Innotech FluorChem Q MultiImage III | Alpha Innotech | Used to visualise ethidium bromide stained agarose gel. | |
| Low Molecular Weight Ladder | New England BioLabs | N3233L | Molecular weight DNA ladder. |
| Vortex Mixer | MidSci | VM-3200 | |
| Mini Centrifuge | MidSci | C1008-R | |
| Dry Bath | MidSci | DB-D1 | |
| NanoDrop 2000C | ThermoFisherScientific | ND-2000C | Spectrophotometer. |
| Wide Mini-Sub Cell GT Horizontal Electrophoresis System | BioRad | 1704469 | Electrophoresis equipment-apparatus to set up gel |
| PowerPac Basic Power Supply | BioRad | 1645050 | Power supply for gel electrophoresis. |
| Agarose | Dot Scientific | AGLE-500 | |
| Mastercycler Gradient | Eppendorf | 950000015 | PCR thermocycler. |
| Centrifuge | Eppendorf | 5810 R | |
| Centrifuge | Eppendorf | 5430R | |
| Wizard SV Gel and PCR Fragment DNA Clean-Up System | Promega | A9281 | |
| Zero Blunt TOPO PCR Cloning Kit, with One Shot TOP10 Chemically Competent E. coli cells | ThermoFisherScientific | K280020 | |
| MyPCR Preparation Station Mystaire | MidSci | MY-PCR24 | Hood dedicated to PCR work. |
| Hamilton SafeAire II fume hood | ThermoFisherScientific | Fume hood. | |
| Beckman Coulter Z1 Particle Counter | Beckman Coulter | 6605698 | Particle counter. For counting cells before plating for RNA extraction. |
| Applied Biosystems Sequence Scanner Software v2.0 | Applied Biosystems (through ThermoFisherScientific) | Software to analyze Sanger sequencing data. |
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