Rusty

Our Christmas present this past year ….

Next stop – Kansas!

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.

RNA is everywhere …

This essay is  follow-up to a previous entry, the second in what may become a series of unpublished but very real results.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

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).

8!

It’s going to be a crazy week …

… here in Kentucky.

Madness

What is a linker histone doing there?

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.

A Saturday in Central Kentucky …

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

Coming attractions

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.

Talks.

Posters.

Enjoy!