cP-RNA-Seq

cP-RNA-Seq

2',3'-cyclic phosphate (cP) RNA sequencing (cP-RNA-Seq) is a method to isolate and sequence RNA species protected by cP at their 3' terminus, which usually prevents adapter ligation. The researchers originally developed this technique to identify transfer RNA (tRNA) species that generate sex hormone–dependent tRNA-derived RNAs (SHOT-RNAs) in human breast cancer cells, but it can be used for other RNA species protected by cP at the 3' end.¹

Total RNA from samples is isolated and gel-purified to the desired length. This mixture contains RNA species with 3' hydroxy, 3'phosphate, and 3'-cP. Phosphatase treatment removes phosphates from the 5' and 3' ends. The RNA is treated subsequently with periodate to cleave the 3'-hydroxyl ends into 2'3'-dialdehydes, while leaving the 3'-cP ends intact. RNA strands with 2'3'-dialdehydes at their 3' terminus are inert to adapter ligation. Strands with 3'-cP are cleaved with T4 polynucleotide kinase (PNK) and ligated to sequencing adapters. After flanking both ends with adapters, the RNA strands are reverse-transcribed, amplified, purified, and sequenced.

Pros:

• Selective isolation, amplification, and sequencing of RNA species protected by cP
• Considerable improvement in efficiency and specificity compared to an earlier method using recombinant tRNA ligase and multiple gel purifications²

Cons:

• False positives will arise when sequencing RNA species with 2'-O-methylribose modifications—such as plant miRNAs, plant and animal short interfering RNAs (siRNAs), and animal PIWI-interacting RNAs (piRNAs)—due to periodate cleavage specificity³

• cP-RNA-Seq: Honda S., Morichika K. and Kirino Y. (2016) Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method. Nat Protoc 11: 476-489
• 1. Honda S., Loher P., Shigematsu M., Palazzo J. P., Suzuki R., et al. (2015) Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci U S A 112: E3816-3825
• 2. Honda S., Morichika K. and Kirino Y. Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method. Nat Protoc. 2016;11:476-489
• 3. Honda S., Morichika K. and Kirino Y. Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method. Nat Protoc. 2016;11:476-489

1. Honda S., Loher P., Shigematsu M., Palazzo J. P., Suzuki R., et al. (2015) Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci U S A 112: E3816-3825