For mRNA molecules, degradation is inevitable, but it may be delayed. One way to extend the lives of mRNA molecules is to give them longer protective tails, which give degradative enzymes something to chew on before they bite into anything vital. The tails can also be made less digestible by ensuring that they are mixed, that is, composed of different nucleotides, not just adenosines (As).
Although it had been known for several years that some mRNA tails are mixed, the protection afforded by mixed tails wasn’t appreciated until researchers at the Institute for Basic Science (IBS) took a closer look at RNA tailing, what is more formally known as “nontemplated nucleotide addition to the 3′ end of RNA.
In 2014, IBS researchers discovered that the mRNA tail is not limited to As. They developed a high-throughput sequencing method, TAIL-seq, to accurately measure the length of poly(A) tails at a genome-wide scale. They found out that other nucleotides other than As, like guanosine (G), uridine (U), and cytosine (C), decorated the tail end. Mixed tails were reported in an evolutionarily broad range of species, including humans, mice, frogs, and fish, adding further importance to this topic.
To extend this work, the IBS researchers decided to clarify the enzymology of RNA tailing. In particular, they found that some of the enzymes that insert As to the tail are able to add also Gs, Us, and Cs, creating a mixed tail. Details of the IBS team’s work appeared July 19 in the journal Science, in an article titled, “Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation.”
“Here we identify TENT4A (PAPD7) and TENT4B (PAPD5) as the enzymes responsible for mRNA guanylation,” the article’s authors wrote. “Purified TENT4 proteins generate a mixed poly(A) tail with intermittent non-adenosine residues, favoring guanosine.”
Interestingly, in cells, Gs are located mainly at the very end of the tail, or at the next to last position. This can be explained by the fact that the enzymes that prune the poly(A) tails stall when they encounter a G, rather than an A, at the end of the tail.
“A single guanosine is sufficient to impede deadenylase CCR4-NOT which trims the tail and exposes guanosine at the 3′ end,” the Science article noted. “Consistently, depletion of TENT4A/B leads to a decrease in mRNA half-life and abundance in cells.”
In other words, the research team found out that a G addition may slow down the trimming of the tail, thereby protecting it.
“Non-adenosines added by TENT4A/B have a considerable stalling effect. Just one is enough to counteract poly(A) trimming enzymes and stabilize the mRNA,” explains Young-suk Lee, one of the authors of the study. The mRNAs with mixed tails were indeed reduced in cells lacking TENT4A/B.
“The mRNA tail has been considered a pure stretch of As with little informational content, except for its length. However, this study proves that even tails of the same length can have a different composition, and a mixed tail degrades more slowly than the pure poly(A) tail,” adds Kim Narry, corresponding author of the study and director of the IBS’ Center for RNA Research. “Through this study, we can understand the life history of complex mRNA by revealing a new kind of mRNA protection mechanism that was not known before.”
In the future, the team would like to expand its research to various biological systems and to understand how misbehaving tailing mechanisms can lead to various diseases. In addition, they would like to develop an RNA-based gene therapy method that utilizes the mixed tailing effect in cells to enhance mRNA stability.