47 days into the new year. 5 grant proposals. What to do to interrupt the tedium?
This essay is follow-up to a previous entry, the second in what may become a series of unpublished but very real results.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 ….
By way of introducing this short entry: as is probably true for most blogs that discuss various and sundry aspects of science, I have tended to focus on reviews or peer-reviewed research papers – “the literature”. There is, however, a whole lot more to the lab than these finished and polished products. What I want to do with this entry is a bit different. Instead of talking about a complete study, I thought I would talk (briefly) about some results from my lab that, for various reasons, never found their way into print. Ideally, someone will read one of these essays and speak up, telling me just what is going on and how it fits in with other data or models.
The following is one such example, a result that is curious and perplexing. I chose it because it comes with pretty pictures, and because it is a segue for another essay that I will post in the future. The data is from a thesis of a student of mine – Kevin Forbes. The experiment itself is 7-10 years old (I have forgotten just when this study was done), and I made sure that Kevin would be OK with this before I posted anything.
The 2011 RustBelt RNA Meeting (RRM) is just around the corner. The venue this year is in Dayton, OH, a short hop. skip, and jump from the Bluegrass. I’ll post a follow-up after the meeting is over, but in the meantime, I though a few links to the talks and posters might be of interest.
The bi-annual Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA Processing is one that I try to attend on a regular basis. The last two meetings (2009 and this year) posed special problems for me, since I am also the driver and mule for Amy’s moving trip to Juniata College. The two institutions – CSHL and JC – don’t seem to “talk” to each other, and move-in has been coincident with the meeting (basically, 1 day apart, not enough time to drive to PA, return, and fly to NY). This means that I have ended up driving from Lexington to Cold Spring Harbor for the past two meetings. Load the car up with a dorm room, drive to Huntingdon PA, unload, and just continue to Long Island.
Well, it turns out that this was a pretty fortuitous choice of travel this year. The 2011 Eukaryotic mRNA Processing Meeting was, as usual, an exciting and productive one. But it may well be remembered as much for the bookends of the meeting – the eastern seaboard earthquake that ushered the meeting in on the 23rd, and Hurricane Irene, that necessitated some creative re-scheduling of the last day and a half of the meeting. Many participants were busier Friday re-scheduling shuttles and flights than listening to presentations. I was able to leave at the crack of dawn Saturday and beat the storm by about half a day.
The bookends aside, the meeting was excellent (as usual). I won’t post specifics here (CSHL has rules about commentary and disclosure that I will give a wide berth to). A few themes do merit mention. One is that polyadenylation and mRNA 3′ end formation was topical this year. This is due largely to studies such as I have discussed here and here. More and more labs have begun to look at alternative polyadenylation in the context of gene regulation and clinical outcomes, and the number of talks and posters that touched on polyadenylation was gratifyingly large.
A second theme was one that has been developing for the better part of a decade. It has become apparent that the various chapters in the life of an mRNA are not separated, either in time or space. The connections between the many steps – transcription initiation, elongation, termination, capping, splicing, polyadenylation, transport, translation, etc. – are being revealed in ever more fascinating detail. This was evident throughout the meeting.
A third theme was technical. In a nutshell, high-throughput DNA sequencing as applied to RNA has become all the rage. Lots of people are using variations on the themes I describe here and here to study alternative polyadenylation. (I hope to be able to discuss additional plant studies in the near future – stay tuned.) This in addition to other RNA-Seq applications, ChIP-Seq, CLIP-Seq, CRAC (see the brief mention near the bottom of this site), and other acronym-encoded approaches. (I’m kicking myself for missing an opportunity to come up with my own clever term. Oh well.) As sequencing becomes more affordable, I think that this trend will continue.
It’s a good day for the Plant Physiology Program at the University of Kentucky, what with two new PNAS papers appearing in the Early Edition almost simultaneously.
One study deals with terpene metabolism in a eukaryotic microorganism:
Niehaus TD, Okada S, Devarrene TP, Watt DS, Sviripa V, Chappell JC. 2011. Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Published online before print July 11, 2011, doi: 10.1073/pnas.1106222108
Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.
The second paper, on a totally different subject:
Wu X, Liu M, Downie B, Liang C, Ji G, Li QQ, Hunt AG. 2011. Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation. Published online before print July 11, 2011, doi: 10.1073/pnas.1019732108
Alternative polyadenylation (APA) has been shown to play an important role in gene expression regulation in animals and plants. However, the extent of sense and antisense APA at the genome level is not known. We developed a deep-sequencing protocol that queries the junctions of 3′UTR and poly(A) tails and confidently maps the poly(A) tags to the annotated genome. The results of this mapping show that 70% of Arabidopsis genes use more than one poly(A) site, excluding microheterogeneity. Analysis of the poly(A) tags reveal extensive APA in introns and coding sequences, results of which can significantly alter transcript sequences and their encoding proteins. Although the interplay of intron splicing and polyadenylation potentially defines poly(A) site uses in introns, the polyadenylation signals leading to the use of CDS protein-coding region poly(A) sites are distinct from the rest of the genome. Interestingly, a large number of poly(A) sites correspond to putative antisense transcripts that overlap with the promoter of the associated sense transcript, a mode previously demonstrated to regulate sense gene expression. Our results suggest that APA plays a far greater role in gene expression in plants than previously expected.
I’ll have more to say about the second paper in another essay. In the meantime, I’m happy to answer questions about it.
(No, Joe and I did not conspire to have these come out on the same day …)