3P-Seq

3P-Seq

Poly(A)-position profiling by sequencing (3P-Seq) is an RNA sequencing method to identify 3' untranslated (UTR) regions in mRNA. Poly(A) selection is used to isolate mRNA from total RNA and biotinylated-splint primers are annealed and splint-ligated to the end of the mRNA poly-A tail. The RNA-primer complex are partially digested by RNase T1, bound to streptavidin, and washed to purify the 3' fragments. Primers corresponding to the 3'-end of the poly(A) tail are annealed and reverse transcribed with deoxythymidine triphosphate (dTTP) as the only dNTP to generate a complementary cDNA strand for the poly(A) tail. The biotin-bound poly(A) RNA fragments are then released by RNase H digestion and purified. P7 and P5 adapters are subsequently attached to the RNA fragment and reverse transcribed to generate cDNA fragments.

Pros:

• Reliable for UTR isoform discoveries
• Prevents internal priming and is specific to 3' ends of poly(A) RNAs

Cons:

• Needs large amounts of RNA
• Technically challenging to perform

• 3P-Seq: Jan C. H., Friedman R. C., Ruby J. G. and Bartel D. P. (2011) Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs. Nature 469: 97-101

1. Hezroni H., Koppstein D., Schwartz M. G., Avrutin A., Bartel D. P., et al. (2015) Principles of long noncoding RNA evolution derived from direct comparison of transcriptomes in 17 species. Cell Rep 11: 1110-1122
2. Jambor H., Surendranath V., Kalinka A. T., Mejstrik P., Saalfeld S., et al. (2015) Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. Elife 4:
3. Spies N., Burge C. B. and Bartel D. P. (2013) 3' UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Genome Res 23: 2078-2090