This essay is follow-up to a previous entry, the second in what may become a series of unpublished but very real results.
For those of you who have downloaded and read Kevin’s thesis, you will have noticed that much effort was expended in characterizing an unusual inhibitor of poly(A) polymerase. Kevin started in my lab in the days before the Arabidopsis genome was completed; in those days, my lab was committed to pursuing biochemical approaches to understand polyadenylation. Unfortunately, it was not possible to observed authentic processing and polyadenylation activity in a plant-derived nuclear extract. (The reasons for this remain unknown, to this day. Quinn Li’s group has succeeded in detecting processing in an Arabidopsis extract, but even in this case the processed RNAs are not polyadenylated by endogenous poly(A) polymerase.) However, it had been reported, in the mammalian and yeast literature, that one or more polyadenylation factor subcomplexes could inhibit the non-specific activity of poly(A) polymerase. The latter is an enzyme that can be detected, assayed, and purified from plant sources. Thus, it made sense to use the inhibition of this activity as a sort of assay for other polyadenylation factors.
This was where Kevin started. Sure enough, he was able to identify a very effective inhibitor of poly(A) polymerase. After ruling out trivial explanations (it wasn’t a nuclease or protease), he pressed on to purify the inhibitor, with the goal of obtaining DNA clones that encoded the inhibitor (or its subunits). One of the components that copurified with the inhibitor was an interesting variant of histone H1.
In addition to showing that the inhibitor was not a nuclease or protease, Kevin tested the efefcts of proteases and nucleases on the activity of the inhibitor. Not suprisingly, it was sensitive to protease treatment; this showed that it is a protein. However, the inhibitor was also sensitive to nuclease treatment, suggesting a role for RNA in its activity. Kevin isolated nucleic acids from the purified enzyme (in those days, this meant phenol extraction and ethanol precipitation), end-labelled anything that came out, and analyze things on sequencing gels. Surprisingly, what he found was a population of one or more small RNA species. The figure from his thesis:
(The inhibitor was called PPF-B.) Because U1 snRNP was known at the time to inhibit poly(A) polymerase, Kevin also did some northerns that showed that the inhibitor was devoid of U1 snRNA. The unknown RNA in the PPF-B preparation was clearly larger than the stable RNAs seen in nuclear extracts (“extract” in the figure), and to this date has not been identified.
There’s more to this story in his thesis. What remains curious (and, as is the case with the connection with H1, incites the overactive imagination) is the association of a small RNA (but NOT an siRNA or miRNA, since it is far too large) with poly(A) polymerase inhibitory activity. Kevin did not follow-up on this, since the “end of the tunnel” for this project seemed years away. So he instead turned his attention to the Arabidopsis ortholog of Fip1p, an effort that resulted in a nice thesis chapter and an interesting JBC paper. The large RNA associated with the inhibitor still lurks in the back of my mind, but so far no obvious explanation has jumped out of the literature. Maybe someone reading this will be inspired to ….