Backdrop and background ….

… 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.

I’ll summarize two of the more interesting findings that are described in the paper.  When we began this work, we expected that a small number of genes would have differences in poly(A) site choice, and that the identification of these genes would lend insight into the phenotypes of the mutant.  (The expectation that few genes would be affected followed from the fact that the mutant grows relatively normally; it didn’t seem likely that wide-ranging changes involving numerous genes would yield such modest affects on growth.)  What we found, however, is that somewhere between 50% and 90% of all genes exhibit differences in poly(A) site choice in the mutant.  This was quite a surprise, and a bit of a disappointment in that the experiment was of no help in identifying genes affected by the mutation and also involved in, say, responses to oxidative stress.  But this finding is also very interesting in that it necessitates a significant re-thinking of our ideas about CPSF30 and mechanisms of alternative polyadenylation.  (One question that we must answer is – how can the plant tolerate such a wide-ranging alteration in poly(A) site choice?)

We also discovered a number of poly(A) sites that were apparent only in the oxt6 mutant.  This was also surprising – we expected that there might be sites that depend entirely on the presence of CPSF30, but we did not expect sites that are masked by CPSF30 (by some unknown mechanism).  More surprising was the additional finding that these sites seem to possess a different polyadenylation signal than the typical plant poly(A) site.  (The results that show this are at the end of this essay.)  These constitute a new class of poly(A) signal that is used only when CPSF30 is removed (or inhibited).   These sites might be a part of the answer to the question posed parenthetically in the preceding paragraph.  But just part of the answer.

There is more to this study, and I am more than happy to answer questions in the comments.  Needless to say, our results have caused us to re-evaluate much of what we thought we knew about CPSF30 and polyadenylation mechanisms in plants.  Given the connection with oxidative stress (as well as other phenotypes that I hope to discuss in a few months), these issues may have considerable practical importance.

Bibliography – CPSF30 in plants

  1. Forbes KP, Addepalli B, Hunt AG. 2006. J Biol Chem 281: 176-186.
  2. Delaney KJ, Xu R, Zhang J, Yun K-Y, Li QQ, Falcone DF, Hunt AG. 2006. Plant Physiol 140: 1507-1521.
  3. Addepalli B, Hunt AG. 2007. Nucl Acids Res 35: 4453-63.
  4. Addepalli B, Hunt AG. 2008. Arch Biochem Biophys 473, 88-95.
  5. Hunt AG, Xu R, Addepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhya A, Dampanaboina L, Marion A, Von Lanken C, Li QQ. 2008. BMC Genomics 9, 220.
  6. Rao S, Dinkins RD, Hunt AG. 2009. BMC Cell Biology 10, 51.
  7. Bell SA, Hunt AG. 2010. FEBS Letts 584, 1449-1454.
  8. Addepalli B, Limbach PA, Hunt AG. 2010. FEBS Letts 584, 4408-4412.


How do the poly(A) signals seen only in the mutant differ from those seen in the wt?  This is illustrated by plotting the relative nucleotide composition as a function of distance from the poly(A) site, for all sites of each class.  When this is done, the wt sites look like:

In this plot, A content is in blue, U in red, C in green, and G in purple.  The important aspect of this is the A-rich region around -25.  (The number of sites that contribute to this plot is given parenthetically above the plot.)

The oxt6-specific sites look like:

Note that there is no A-rich region for these sites.

13 Responses to Backdrop and background ….

  1. Ed Darrell says:

    Nice to see the blog back in action!

  2. Arthur Hunt says:

    I forgot to mention – the first author of this study is a high school teacher who works in south central Kentucky, at Franklin-Simpson High School. He wrote the program we used to compare poly(A) site distributions.

  3. Ed Darrell says:

    That’s cool. Cynic that I am, I gotta ask: Did his district punish him yet?

    More hopefully, can he continue research, and does his district support him at all?

  4. Clem Weidenbenner says:

    Have to echo Ed’s welcome back…. Rusty is darling, but I was so looking forward to some new content.

    Just got a copy of the article (BTW, thanks for going OA). Will be paying special attention to the environments where oxt6 and WT are compared. Without much more than a hunch I’m suspecting the unexpected phenotypic similarities seen might unravel if more stressful environments are applied.

    Do you have any feeling on the issue of dosage? Would a heterozygote (say the F1 of an oxt6 x WT) favor one parent or the other for polyA site choice?

  5. Arthur Hunt says:

    Ed, I don’t think I want to know (about punishment).

    Kidding aside, I’m hoping to keep working with Patrick. There are a couple of possibilities that we will look into. His district, as you might imagine, is probably limited for resources (although it let him obtain a surplus thermocycler). But a few hundred dollars of stuff (some Taq polymerase, a few primers, and some reagents to purify DNA) would go a long way as far as setting up some lab exercises. That’s something I can swing, so ….

  6. Arthur Hunt says:

    Hi Clem,

    Thanks for the comment. You ask a very good question about dosage. To be honest, we hadn’t thought about doing deep sequencing with a heterozygote. We know that the heterozygote is phenotypically indistinguishable from the wild-type. (The mutant has many interesting phenotypes, only one of which has been published. We’re working on a manuscript that deals with some more characteristics.) We also have a battery of lines that are the oxt6 background and express varying levels of transgenes that encode CPSF30. There seems to be no differences in these lines as well. These experiments are also being written up.

    The comment about stresses is also very insightful. There are many ideas along these lines that we have had, and we are still sorting out what directions will be the most fruitful. Hopefully, some funding agencies will agree with our decisions.

  7. bergounioux says:

    Dear Colleague,
    CPF30 has a spliced form? This form is not mentioned in your manuscript? Could you differentiate the two forms in your study?
    C. Bergounioux

  8. Arthur Hunt says:

    (Dr?) Bergounioux,

    Thanks for your interest in this essay and in the subject of the study. The gene that encodes CPSF30 in plants yields two different mRNAs. One in turn may be translated to yield the 250 amino acid CPSF30, while the other yields a larger (>650 amino acid) polypeptide. The smaller protein has been extensively studied, as the publication list at the end of the essay indicates. We don’t know much about the larger protein. We are studying oxt6 plants that express different forms of CPSF30 (small, large, etc.) and will hopefully have an interesting story to tell in the future.

    More about the two CPSF30 mRNA isoforms in plants can be found in reference 2 and in a recent paper that I plan on discussing on this blog in the near future.

  9. bergounioux says:

    Dr Hunt,
    Thank you very much for your answer. Please could you dissociate the calmodulin binding to the RNA binding for the complementation? Is the 30M mutant able to complement oxt6?

  10. Arthur Hunt says:

    Hi Catherine,

    To answer your questions:

    1. We can indeed dissociate the calmodulin binding from the RNA binding. The 30M mutant you mention does not bind calmodulin, but it does bind RNA as well as the wild-type protein, as far as we can tell. This is good since it helps with the complementation experiments you suggest.

    2. We are currently studying oxt6 lines that express wild-type CPSF30 as well the 30M mutant. Briefly, the 30M mutant does complement some of the mutant phenotypes but not others – a paper on this is in the works. We have some high throughput sequencing data for these plants, but we may need more data before we can say anything definitive about the polyadenylation phenotypes of the various lines.

  11. Balu says:

    It is heartening to see a good amount of interest in these research findings.
    Wish you all a Peaceful, Productive and Reflective New Year!

  12. […] This list raises many questions. For example, CPSF30 is one of two proteins that cooperate to bind the AAUAAA polyadenylation signal in mammals (see the pictures at the end). Plants have an analogous motif (we call it the Near Upstream Element, or NUE), and it seems to be utilized just fine in plants that do not make CPSF30. To be sure, most poly(A) sites that require CPSF30 also have the typical plant NUE, and most sites that are seen only in CPSF30 mutants do not have the NUE. But it seems clear that general NUE usage happens just fine in the absence of CPSF30. (I have talked about polyadenylation signals here, and the CPSF30 mutant here.) […]

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