… for the recently-started Spring Meet at Keeneland (it really is this pretty), I suggest that you find time to attend the 2013 Naff Symposium at the University of Kentucky. This is an annual event put on by the Dept. of Chemistry and centers on aspects of chemistry and molecular biology. This year’s topic is The Origin of Life, and the line-up of speakers is pretty amazing.
47 days into the new year. 5 grant proposals. What to do to interrupt the tedium?
… was it? So bad that my mother called me immediately after the game. Not to console me, but to gloat.
(What game? If you have to ask, then …)
This is the question I think about a lot, and one I spent a some time on in a recent minireview. The answer is, in a nutshell, very.
One of the things I had to do for this review was try and make sense out of the different approaches that have been described recently for studying alternative poly(A) site choice in plants. One of these – the use of high-throughput sequencing to sequence cDNA tags that query the exact mRNA-poly(A) junction – has been discussed previously, in a general sense and in terms of a study of poly(A) site choice in plants. In the latter study, it was determined that about 70% of plant genes possess at least two poly(A) sites.
… for a recent paper. And a short summary as well.
I know it seems like a long time since my last entry here, and it has been. This is what happens when one makes promises to one’s self.
Back in late May, I was looking forward to adding an entry describing a new paper that was (in my optimistic eyes) on the verge of being accepted, and I told myself that this entry would be the next one on this blog. As one might imagine, reality stepped in, and acceptance of the paper was delayed by quite awhile. But this changed recently, and on Nov. 6 the study was finally published online. (Coincidence with the outcome of the Presidential election? Who knows.) So I can pick up where I left off several months ago.
Two different lines of research led to the current study. My lab has been studying a particular subunit of the plant polyadenylation complex – CPSF30 – for some time. A list of our publications on this protein is at the end of this essay. What was of particular interest were two findings. One of these was that the Arabidopsis CPSF30 could bind calmodulin, and that the RNA-binding activity of the protein was inhibited by calmodulin in a calcium-dependent fashion. The second finding (discussed previously on this blog) was that two of the cysteine residues in one of the zinc finger motifs of the protein were engaged in a disulfide linkage; reduction of this bond inhibits another biochemical activity of the protein (this finding was described in Addepalli and Hunt, 2008). What is interesting about these findings is that these mechanisms or pathways (calmodulin and a reducible disulfide bond, respectively) that connect with cellular “sensory” pathways both inhibit the plant CPSF30. Putting things simply, these studies raise the possibility that CPSF30, and thus polyadenylation, may be directly regulated by cellular signaling systems in plants.
The second line of research may be traced to a fortuitous finding in Deane’ Falcone’s laboratory. (Deane was in the Department of Agronomy here at the University of Kentucky when he made this discovery; he since moved on to the Dept. of Biological Sciences at UMass Lowell.) This finding was described in a 2008 paper in PLoS ONE; briefly, Deane found that an Arabidopsis mutant with a T-DNA insertion in the gene (OXT6) that encodes CPSF30 had a greater tolerance than the wild-type to different sorts of oxidative stresses.
The inhibitory effects of calmodulin and sulfhydryl reagents (that reduce the disulfide linkage mentioned above) on CPSF30 in vitro raise the possibility that activation of calmodulin in the cell, or altering the redox status of the cell, might inhibit CPSF30 in vivo. This could lead to some sort of change in polyadenylation in the cell, change that would be manifest in altered growth or responses (such as, say, to oxidative stress). Thus, an obvious question to ask is – how does the inhibition of CPSF30 affect polyadenylation?
The availability of the oxt6 mutant allowed us to ask this question. Briefly, we reasoned that mutational elimination of CPSF30 might approximate the inhibition of the protein; thus, a characterization of the oxt6 mutant should provide insight into the consequences of inhibition of CPSF30 via calmodulin or redox mechanisms. To tackle this, we turned to the high-throughput poly(A) site methodology that we developed and described in 2011, using the approach to assess differences in poly(A) site choice between the mutant and wild-type. Basically, we used the high throughput approach to determine poly(A) sites for most expressed genes in the wild-type and oxt6 mutant. This is the study that was recently published.
This weekend past was spent in Huntingdon, PA (about 25 miles from State College) attending Amy’s graduation from Juniata College. Amy earned a B.A. with a major in History and minor in Anthropology. She’ll be pursuing a Master’s degree at the University of Kansas, majoring in African Studies. This interest is, to a large extent, an outcome of her most excellent semester in The Gambia.
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.
Wrapping up a busy semester in Lexington with a leisurely Saturday …