mRNA Alternative Polyadenylation Lifestyles Comes into Focus

As the only epigenetics media group based out of southern California, we’ve learned to be very accepting of alternative lifestyles, which is why this method caught our eye. mRNA are a diverse bunch, with all kinds of alternative polyadenylation that can affect their cellular localization and stability. RNA Seq has enabled unprecedented glimpses into the transcriptome, but most RNA Seq protocols fall short on nailing down mRNA poly-A tails.

Well, a clever team of researchers from Germany claim that their new method for mapping poly(A) tails will have your sorting through these sites in no time. The method is called 3’T-fill, and it outperforms existing techniques, like RNA-Seq, with the added bonus of being quantitative.

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The German team fills in the poly(A) stretch in mRNAs with dTTPs. Sequencing then starts after the poly(A) sites and goes into the 3’ untranslated regions. This gets around the problem of low-quality sequences you sometimes get from protocols that read through the poly(A)s, which might happen because of polymerase slippage or mispriming.

They say that 3’T-fill outperforms competing techniques and offers several advantages:

  • With 3’T-fill, the researchers fragment the RNA then reverse transcribe it, avoiding delicate RNA handling.
  • They also bind the fragments early on to magnetic beads. That means they can easily scale up and automate the process.
  • Because 3’T-fill doesn’t sequence 5’ into the tail, you don’t have to do size selection and you don’t have to do any trimming.
  • It’s the easiest and most efficient of the methods they compared head-to-head.
  • You only wind up with one read for each transcript, regardless of how big the transcript is. That makes it more quantitative than, say, RNA-Seq.

The researchers used 3’T-fill on yeast mRNAs and found that even in genetically identical populations, the mRNAs can be different.

They also say that you could adapt the method to work on non-poly(A) RNAs, like bacterial ones.

Don’t miss a thing—catch the details at Nucleic Acids Research, January 2013.