Backdrop and background ….

November 9, 2012

… 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.

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Rusty

May 22, 2012

Our Christmas present this past year ….

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Next stop – Kansas!

May 15, 2012

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.

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RNA is everywhere …

May 4, 2012

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 ….


Fear and polyadenylation

April 17, 2012

No, this post is not about the fear that our favorite subject strikes in the minds of students who are struggling with concepts and principles of gene expression.  Rather, it’s about an interesting story that helps to illustrate (as if this is needed) the relevance of polyadenylation (and specifically poly(A) site choice) to medical science.

Mention has been made on this blog of a correlation between poly(A) site choice and cancer.  Many meta-analyses and high throughput sequencing studies have also noted a related phenomenon – a great deal of alternative polyadenylation that seems to be specific for neural cells and tissues.  One example of this is recalled in a recent paper that suggests a link between an alteration in alternative polyadenylation and aspects of memory and anxiety in mammals (including humans).

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8!

April 3, 2012

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It’s going to be a crazy week …

March 25, 2012

… here in Kentucky.


Madness

March 14, 2012


What is a linker histone doing there?

March 3, 2012

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.

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A Saturday in Central Kentucky …

December 3, 2011

Wrapping up a busy semester in Lexington with a leisurely Saturday …

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