Q: What is RNA tailing?
A: Polyadenylation, commonly known as poly(A)-tailing, is the process of adding a long chain of adenine nucleotides (poly(A) tail) to the 3’-end of a messenger RNA (mRNA) molecule. This modification occurs during RNA processing in eukaryotic cells and serves several essential functions:
- Stability: The poly(A) tail increases the stability of the mRNA molecule, protecting it from degradation.
- Transport: It aids in the export of the mRNA from the nucleus to the cytoplasm.
- Translation: The tail enhances the translation efficiency of the mRNA into proteins by facilitating the binding of ribosomes.
This process is crucial for proper gene expression and regulation in cells.
Q: How does enzymatic poly(A)-tailing compare to, or differ from, co-transcriptional tailing methods?
A: Co-transcriptional poly(A)-tailing is accomplished by encoding the tail sequence in the DNA transcription template. Thus, as the in vitro transcribed (IVT) RNA is made by the RNA polymerase, the tail is also made as part of the runoff transcript. The benefit to this approach is that in a single enzymatic reaction, poly(A)-tailed RNA can be generated as opposed to just non-tailed RNA. The drawback to this approach is that the size of template-encoded tails is limited to ~60-A’s. While longer poly(A) tails can be cloned into transcription templates (e.g., 120-A’s) such clones are unstable in E. coli and will condense back down to a stable state of ~60-A’s long after multiple passages of the bacterial strain.
Post-transcriptional poly(A)-tailing involves enzymatic treatment of the IVT RNA with Poly(A) Polymerase. The benefit to this approach is that one can generate tail lengths of any desired size. For mRNA expression in eukaryotic cells, the longer the poly(A) tail is, the better the expression. Natural, in vivo protein-expressing mRNAs commonly have poly(A) tails of 150-200-A’s in length. Some mRNAs have shown an additional expression boost with poly(A) tail lengths of 300-A, 400-A and longer.
