As we think of gene regulation from a cis perspective, we are driven to focus our attention on what we call regulatory elements. These are typically short DNA or RNA sequences, which blend together to instruct a gene about its output. A site for the binding of a transcription factor, or of a microRNA, for example. They are more or less the same from the perspective of the trans regulator, but it is their combination that gives a specific gene the instructions to work within a cell: up, down, here, there, more protein, less protein, and so on.
When we started this project, we were fascinated by the idea that elements such as poly(A) tails might add something unusual to this logic of gene regulation. First of all, they can be quite long. Second, their length can change drastically during the life of an mRNA. And third, they are stretches of As, but we know that sometimes they can be terminally modified. In short, they are dynamic.
So much is known about them, and yet, as often in biology, we do not really get how this stuff works. They are important for translation and degradation, they are heavily shortened or lengthened but their length does not seem to correlate with any translation or turnover rate, unless we look at specific biological processes. When we approached these problems, it turned out that there were very few methods for studying tails in a systematic way, and as we know, all methods come with pros and cons. In this case the unsolved problems were mainly two: I) we could not sequence through the tails, but just measure their length, and II) we could just assign a tail to a gene via a little chunk of sequence that is attached to it. For reasons that I am not listing here, we thought that single-molecule real-time sequencing would be a good way of solving these issues, if we were able to construct cDNA libraries in a way to feed entire mRNAs, including the tails, to a PacBio machine. We therefore teamed up in the lab and tried to make it work. It was hard, and now we finally have FLAM-seq. I must say that we are really happy with the method, please try it! Now we can get, for hundreds of thousands of transcripts, the entire mRNA sequence and the entire tail sequence in one shot, and that is really exciting.
We already found interesting, unexpected things, which I leave to the paper (you can read it here: https://www.nature.com/articles/s41592-019-0503-y), but the real fun starts now. There is still so much to understand about poly(A) tails and gene regulation. We are already working on some cool follow-ups, so stay tuned!
Post written by Ivano Legnini. FLAM-seq results from a great collaboration with Jonathan Alles, Nikos Karaiskos, Salah Ayoub and Nikolaus Rajewsky.