On the functional significance of alternative splicing

May 29, 2009

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.

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More gene targeting in plants

May 25, 2009

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

May 25, 2009

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.

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Guidelines for quantitative PCR

May 25, 2009

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.

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One year later …

May 25, 2009

Today is the one year anniversary of the beginning of this blog.  I wasn’t sure how things would develop, and still am not.  We’ve had a few good discussions, but what I have noticed most of all is how I get a steady stream of hits on the polyadenylation background essays.  It’s enough to imagine that this blog is sort of an alternative to wikipedia for those looking for information about this process.  This is good, but this interest has not come with questions.  Either these essays are clear (not likely), or someone out there is being shy.  So, those readers who are using this blog as a vehicle to learn about the process, don’t hesitate to ask questions.  You obviously want to learn something about the subject, and the best way to learn is to ask.

Graduation week

May 14, 2009

I spent the last week traveling to two graduations.  Our oldest daughter graduated on Monday from the College of Wooster, with a BA in Geology.  Heather will be attending the University of Montana in pursuit of a Master’s Degree.

Earlier, on May 6, my sister’s oldest son (Corey) graduated from Officer Candidate’s School at the US Coast Guard Academy in New London, CT.

Ensign McPartlin.  That has a great sound.

A few photos beneath the fold. Read the rest of this entry »

Where polyadenylation, siRNAs, and DNA methylation meet

May 14, 2009

It has become more apparent in recent years that the different aspects of gene expression – transcription initiation, transcription elongation, mRNA capping, splicing, and polyadenylation, transport of the mRNA to the cytoplasm, translation, and mRNA quality control – are rather extensively interconnected.  One corollary is that the polyadenylation complex, through various of its subunits, plays roles in various of these other processes.  This has been established for the most parts in mammalian and yeast models, but some recent work in plants is adding new and important variation to this theme.

A most recent of such studies has appeared online on PNAS.  This study, from the lab of Caroline Dean, reveals that the polyadenylation factor subunit FY (a homolog of the yeast protein Pfs2), acting in concert with the flowering regulator FCA, plays a crucial role in chromatin modifications that regulate the expression of the FLC gene.  Interestingly, this effect is not limited to just the FLC gene. Rather, other genes that are silenced by small RNA-mediated DNA methylation also require FY for this silencing.  This provocative finding seems to place FY in some sort of proximity to the small RNA-guided DNA methylation machinery, and may have some relevance to many aspects of transcription and mRNA quality control.

The abstract and citation follows. As always, enjoy. Read the rest of this entry »