Putting the polyadenylation complex together

One of the reasons for the slow pace of entries here has been the intrusion of, um, life into my life.  (Yeah, figure that one out.)  Earlier this summer, a paper from my lab was published in BMC Cell Biology.  Seeing as it’s Open Access, and since it has a bit of relevance to a theme introduced in this essay, I thought I would point it out here.

The story in a nutshell – one of the subunits of the polyadenylation complex is the so-called CPSF30 protein, or its yeast relative Yth1.  (Yth1 looms large as one of the few subunits in the Giardia complex.)  What Drs. Suryadevara Rao and Randy Dinkins did was study the places within the cell where CPSF30 goes, and what happens when one co-expresses this protein with other polyadenylation complex subunits.  They did this by attaching the various proteins to fluorescent proteins and following the fusion proteins using microscopic techniques.

The results corroborated other studies that detailed interactions between various of these proteins.  However, a rudimentary deletion analysis showed that these interactions by and large involve parts of CPSF30 that are not found in the mammalian or yeast proteins.  Since the CPSF30 interacts with the other proteins of interest in this study (the 160, 100, and 73 kD subunits of the cleavage and polyadenylation specificity factor, or CPSF) in other eukaryotes, it stands to reason that the interactions themselves must have evolved independently.  This in turn suggests a somewhat different trajectory in the evolution of the complex in different eukaryotic lineages.  It also raises the possibility that the different complexes may process and polyadenylate RNAs in subtly different ways.

Some pretty pictures and a link to a fascinating movie may be found beneath the fold.  Enjoy.

Four weeks on the road …

Well, not all on the road.  Most of one week at home packing between trips, and a few days at Cold Spring Harbor in NY.

Hunt Lab Reunion

… at the 2009 Cold Spring Harbor Meeting on Eukaryotic mRNA Processing.  From left to right, Kevin Forbes (former Ph.D. student), yours truly, and Quinn Li (former Ph.D. student).

The mini-reunion was a highlight of a great meeting.   I may (no promises!) post a brief summary in a few days, once I figure out how to summarize things without violating the spirit of the meeting (what happens at CSH stays in CSH).

Whew!

The past few weeks (it seems a lot longer) have been spent largely on the road.  The task has been to move my oldest daughter (Heather) to Missoula MT, where she will be attending the University of Montana.  We folded a family road trip into the move and took a few weeks to accomplish the task.  A brief photo recap is beneath the fold.  I hope to return to some of the more usual blog entries after the next road trip (moving Amy back to Juniata, wrapped around the Eukaryotic mRNA Processing meeting at the Cold Spring Harbor Laboratory).  Enjoy. (Warning – quite a few pictures, which may make for slow loading.)

NAPC followup

Professor Steve Steve wil NAPC panelists RIchard Hoppe, Jason Rosenhouse, and yours truly.

Late last month I participated in a panel discussion on “Confronting Creationism” as part of a day of outreach/education activities associated with the 9th North American Paleontological Convention in Cincinnati.  I was joined on the panel by Richard Hoppe and Jason Rosenhouse.  What follows are a random string of comments and recollections from the day’s events (I’m not a reporter and didn’t take notes; Jason has posted on the meeting at more length).

There’s more to UK …

That would be UK as in the University of Kentucky.

We’ve been splayed all over the news for the past several months, embarrassingly so.  Seems like we stole the show during and shortly after the NCAA tournament, and our new coach deliberately or inadvertently has kept us on p1 for much of the past several months.  It wouldn’t surprise to think that, to the rest of the country (and world), the University of Kentucky was the name of some sports franchise.

Well, the president of UK has ’bout had enough of this.  His editorial in today’s Lexington newspaper was refreshing in many ways – a slap in the H-L’s face, a grasping of the readership by the collar and shaking some sense into them, a rather public display of annoyance and exasperation, and yes, an implicit plea to taxpayers to support his school.  UK is more than 12 (yup, JM isn’t coming back) athletes who ply their trade for five months of the year.  It’s good to see the president remind the Commonwealth of this.

On the functional significance of alternative splicing

Alternative splicing – the choice of different splice sites and/or exons in a primary transcript that possesses numerous exons and introns – is a widespread phenomenon.  With the advent of very sensitive as well as high-throughput techniques, it has proven possible to identify alternatively-spliced transcripts for many, perhaps a majority, perhaps all genes.  However, the very sensitivity of the techniques raise the interesting and important question of the functional significance of what is observed.  Thus, it is possible that much (most, all?) of the alternatively-spliced mRNA isoforms are the results of splicing errors.  (Some in the blogosphere are of the opinion that alternative splicing is mostly artifact.)  Accordingly, studies that speak to the functions of the products of alternative splicing are always of interest.

A recent study from Stephen Mount’s lab illustrates an excellent approach to this problem.  In this study, two different isoforms of a so-called SR protein (the Arabidopsis SR45 splicing factor) were studied.  These isoforms are encoded by different alternatively-spliced mRNAs, and differ by eight amino acids that correspond to one of two 3′ splice sites that are chosen in the course of pre-mRNA processing.  Loss-of-function mutant plants that do not make SR45 show a range of developmental phenotypes that affect flowers and roots.  Interestingly, when one isoform is expressed* in a loss-of-function mutant background, the flower phenotype is reversed but not the root phenotype.  Conversely, expression of the other isoform restores normal root growth but not flower morphology.  The bottom line is that the two SR45 isoforms have distinct functions.  Thus, at least in this case, alternative splicing has important roles in growth and development.

More gene targeting in plants

A follow-up to an entry I made a few weeks ago, showing that engineered zinc finger nucleases can be used to target gene insertion in maize.  Abstract and citation without commentary.  Enjoy.

Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable1, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus2. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.

Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD. 2009. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437-441.

Alternative polyadenylation in development

One of the things that is an open book is the true scope and physiological relevance of alternative polyadenylation.  A recent report in PNAS stirs this pot a bit (even if it leaves things still very much up in the air).  Briefly, this group has analyzed various large-scale gene expression repositories – ESTs, SAGE, and microarray – and found a tantalizing possible progression of 3′-UTR length during development.  Specifically, it seems as if global (or average) 3′-UTR length increases during the course of embryogenesis.  This change in the length of 3′-UTRs seems to be due to differential poly(A) site choice.  As I said, very tantalizing.

The abstract and brief commentary follows after the fold.

Guidelines for quantitative PCR

One of the more vexing and difficult issues in molecular biology is the use of PCR as a quantitative assay.  It is easy to find a wide range of approaches to this, and even easier to find papers that refer to “semi-quantitative” PCR.  As one might expect, there is a range of quality in the literature when it comes to these assays.  Worse, though, is the paucity of information that is often provided – things like the numbers of replicates, statistical tools used to analyze the data, and the like.  THis makes it hard to follow many studies, and to replicate the work of others.  (Needless to say, reviewing these papers is a bear.)

There has been a mini-debate of sorts in The Plant Cell over the past few years, and a recent issue has two additions to the discussion.  Follow below the fold for more.