A good day

July 11, 2011

It’s a good day for the Plant Physiology Program at the University of Kentucky, what with two new PNAS papers appearing in the Early Edition almost simultaneously.

One study deals with terpene metabolism in a eukaryotic microorganism:

Niehaus TD, Okada S, Devarrene TP, Watt DS, Sviripa V, Chappell JC.  2011. Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii. Published online before print July 11, 2011, doi: 10.1073/pnas.1106222108

The abstract:

Botryococcene biosynthesis is thought to resemble that of squalene, a metabolite essential for sterol metabolism in all eukaryotes. Squalene arises from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene. In principle, botryococcene could arise from an alternative rearrangement of the presqualene intermediate. Because of these proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence isolated squalene synthase-like genes from Botryococcus braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encodes for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when combinations of the synthase-like enzymes were mixed together, in vivo and in vitro, robust botryococcene (SSL-1+SSL-3) or squalene biosynthesis (SSL1+SSL-2) was observed. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved interdependently for specialized triterpene oil production greater than 500 MYA. Coexpression of the SSL-1 and SSL-3 genes in different configurations, as independent genes, as gene fusions, or targeted to intracellular membranes, also demonstrate the potential for engineering even greater efficiencies of botryococcene biosynthesis.

The second paper, on a totally different subject:

Wu X, Liu M, Downie B, Liang C, Ji G, Li QQ, Hunt AG. 2011. Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation. Published online before print July 11, 2011, doi: 10.1073/pnas.1019732108

The abstract:

Alternative polyadenylation (APA) has been shown to play an important role in gene expression regulation in animals and plants. However, the extent of sense and antisense APA at the genome level is not known. We developed a deep-sequencing protocol that queries the junctions of 3′UTR and poly(A) tails and confidently maps the poly(A) tags to the annotated genome. The results of this mapping show that 70% of Arabidopsis genes use more than one poly(A) site, excluding microheterogeneity. Analysis of the poly(A) tags reveal extensive APA in introns and coding sequences, results of which can significantly alter transcript sequences and their encoding proteins. Although the interplay of intron splicing and polyadenylation potentially defines poly(A) site uses in introns, the polyadenylation signals leading to the use of CDS protein-coding region poly(A) sites are distinct from the rest of the genome. Interestingly, a large number of poly(A) sites correspond to putative antisense transcripts that overlap with the promoter of the associated sense transcript, a mode previously demonstrated to regulate sense gene expression. Our results suggest that APA plays a far greater role in gene expression in plants than previously expected.

I’ll have more to say about the second paper in another essay.  In the meantime, I’m happy to answer questions about it.

(No, Joe and I did not conspire to have these come out on the same day …)


The Next Generation?

February 28, 2011

Not Star Trek.  What we’re talking about here is DNA sequencing, and the impact it is having, and will have, on studies of polyadenylation.

Since last summer, there has been a spate of papers describing the application of so-called Next Generation DNA sequencing (in its many manifestations) to the matter of polyadenylation.  The general idea is simple – to generate and analyze large numbers of short DNA tags that are derived from the junctions of the poly(A) tail and bodies of mRNAs.  The expected outcomes of such studies are qualitative and quantitative descriptions of the genome-wide distributions of polyadenylation sites.  This information would help to better annotate (or describe) the genome, and to help identify unusual occurrences.  The latter might include alternative poly(A) sites, sites associated with as-yet unidentified transcripts, and sites that define antisense RNAs.

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Might this apply?

December 24, 2010

A recent article in RNA – “The exozyme model: A continuum of functionally distinct complexes” – provides at once a timely review of exosome structure and function, and an interesting hypothesis that attempts to explain some interesting features of the exosome as it is found in different eukaryotes.

Recall that the exosome is the term for a (THE) RNA degrading machine in eukaryotes, and that it is analogous in many ways to the degradosome in bacteria.  Over the years, various and sundry exosome subunits have been implicated by genetics or biochemistry in numerous RNA processing and degrading events or systems.  However, there are differences, in terms of subunit composition and activity, between different organisms.  Because of these differences (that I won’t list here – Kiss and Andrulis do an excellent job that would take thousands of words to summarize), the authors of the cited review propose that the “exosome” is better thought of as a collection of “exozymes”, all of which share some subset of the subunits that collectively are usually associated with the conceptual exosome. In the authors’ own words: Read the rest of this entry »


Cleveland Rocks!

October 30, 2010

Not just the Rock and Roll Hall of Fame.  Last weekend, a group of midwestern RNA scientists gathered for the annual Rustbelt RNA Meeting in Cleveland. (There’s a clever pun hidden in the name, one that may fall by the wayside in the next year or so.)

Here is a link to the abstracts.  So readers can take a peek into just what excites RNA scientists.  Enjoy.

PS – just out of curiosity, does the name “Rustbelt” carry negative connotations for readers here?  Just wondering.


I didn’t know it could do this ….

October 8, 2010

What better way to cut short this unanticipated hiatus (no real reason – just got busy with other stuff) than point readers to a new study that reveals yet another unexpected and unusual property of a plant polyadenylation factor subunit.  In a nutshell (which is posted, as the abstract, beneath the fold), Balu Addepalli has found that two of the cysteines in one of the three CCCH zinc finger motifs in the Arabidopsis CPSF30 protein (that’s a mouthful) are actually engaged in a disulfide bond.  This was unexpected (to me, at least) because it is usually assumed that the cysteines and histidine residue coordinate around a zinc ion (hence the term zinc finger).  As indicated in the abstract, this new finding raises the possibility that the Arabidopsis CPSF30 is regulated in part by redox control, through oxidation and reduction of the disulfide bond.

So, this adds one more item to the cart of novelties regarding the plant CPSF30.  The gene encoding the protein is alternatively spliced, such that the CPSF30 part of the resulting protein is fused to a novel, poorly-understood domain.  (See this article for the details.)  The protein is not essential for growth (in contrast to its yeast counterpart, Yth1p).  CPSF30 mutants (we call them oxt6 mutants) are tolerant to oxidative stress.  (This may be relevant to the disulfide linkage story – future research will tell).  The Arabidopsis CPSF30 is an endonuclease, an activity for which a function has not been firmly established (although my group thinks we know – see this study).  This protein is also a calmodulin-binding protein, which raises interesting possibilities concerning regulation, signaling, and polyadenylation. Read the rest of this entry »


CFIm – even more reducibility

July 23, 2010

Earlier, I discussed interesting results indicating that the canonical Cleavage and Polyadenylation Specificity Factor (or CPSF) functions in the 3′ processing of histone mRNAs, a reaction that involves a subset of the proteins associated with mRNA 3′ end formation and polyadenylation. One developing idea I mentioned was that the polyadenylation complex may consist of a core that functions in other processes (such as histone mRNA 3′ end formation), and that its activity is modulated by a varying suite of accessory factors.  Recently, a study appeared that elaborates on this finding.  Briefly, Ruepp et al. have found that the 68 kD subunit of the so-called mammalian Cleavage Factor I (CFIm68, for short) associates with the U7 snRNP complex, and functions in histone mRNA 3′ processing.  As interestingly, they report that this subunit does not act with its partner in the nuclear polyadenylation complex, the 25 kD subunit of CFIm (CFIm25). Read the rest of this entry »


Cans of worms …

June 12, 2010

Two recent reports from the Pubmed wires:

One report describes a global analysis of 3′-UTRs in C. elegans.  This group collected information on 3′-UTRs at different stages of development of the organism; this information came from data mining, cDNA and 3′-RACE sequencing, and 454 sequencing of 3′-end tags (to briefly summarize the approaches – read the paper to get a better flavor).  There is lots of information in the paper, but two things revisit issues that have been discussed in this blog.  One matter is that there is extensive alternative polyadenylation, such that mRNAs early in development are shorter longer than those later in development.  This is because of extensive alternative polyadenylation, and the result is recalls findings from similar studies in humans. It would seem as if, in animals at least, there is a global and important shift in poly(A) site choice during development.  The mechanism(s) underlying this shift remain an open question.

Second, there is no canonical poly(A) signal for many of the alternative polyadenylation sites that these authors see.  This is also similar to what is seen in humans, and recalls the more general themes of poly(A) signals that are discussed in this essay.  While no specifics can be stated, this suggests that the polyadenylation apparatus early in development is different, or that it is modified or regulated such that it has a different set of RNA sequence preferences.  It will be fascinating to see how these questions sort out.

A second report describes an interesting genetic screen that implicates a C. elegans homolog of the yeast polyadenylation factor subunit PFS2 in neural development. (Sorry for the link to Pubmed – the journal doesn’t yet have the link up.  Also, obviously, I am going by the abstract here.  If the paper raises additional issues, I will update this essay appropriately.)  This is interesting because the plant homolog of Pfs2, FY, has important regulatory functions in flowering and in chromatin-mediated gene silencing.  This raises the interesting (but highly speculative) possibility of an evolutionarily-conserved function for FY/PFS2.  It will be interesting to see if the C. elegans homolog plays analogous roles in chromatin modification.

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Alternative polyadenylation?

June 3, 2010

A recent study of transgene expression in plants revisits themes that recur in the literature every so often.  Namely, that “alternative polyadenylation” and low-level transcription well beyond a plant polyadenylation signal are common occurrences in plants.  These phenomena are not limited to just transgenes, but are seen with most (if not all) genes.

Why the scare quotes?  Because, while the events documented in this study are formally occurrences of alternative polyadenylation, they reflect the inherent 3′ end microheterogeneity that is seen in almost all plant genes, and probably involves subtly different handling of a single polyadenylation signal.  This sort of poly(A) site heterogeneity should be distinguished from the occurrence of clearly distinct polyadenylation signals, separated by hundreds or thousands of nucleotides along a transcript.

In any case, this short report reinforces the notion that transcriptional readthrough from transgenes can impact the expression of “host” genes via posttranscriptional gene silencing.

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It’s getting crowded

May 1, 2010

One of the interesting things about the polyadenylation complex is the multiplicity of RNA-binding proteins in the system.  As I mentioned before, there are many such proteins, more than the numbers of cis elements typically associated with polyadenylation.  Closer inspection of the complex reveals more paradoxes involved RNA-binding proteins and polyadenylation.

Recently, a rotation student in my lab published a study that reported the discovery of a new RNA-binding domain in the plant polyadenylation complex (1).  There are two findings that are described this study.  The obvious one, that can be extracted from the title, is that the 77 kD subunit of the Arabidopsis Cleavage Stimulatory Complex (CstF77) is in fact an RNA binding protein.  In this report, the new RNA-binding domain was localized to the C-terminus of the protein.  This is interesting because this part of CstF77 is not conserved in its eukaryotic (yeast and mammalian) counterparts.  This raises the possibility that RNA binding by CstF77 is a plant-specific phenomenon.

The second interesting finding is the location of the RNA-binding domain. The structures of two eukaryotic CstF77 proteins have been solved, and  it has been established that the protein exists as a dimer (2,3).  The dimer is “held together” by extensive interactions involving the “middle” of the protein, and several of the characteristic “HAT” motifs (4) in the protein.  The C-termini of each subunit of the dimer probably extends into the cavity formed by the dimer, as suggested in the figure below.

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The importance of poly(A) site choice

January 1, 2010

To ring out 2009, a brief mention of some studies that reinforce the conclusions mentioned in this essay.  The basic theme – it looks more and more as if poly(A) site choice can vary for many, many genes in humans, and that a general trend towards shorter 3’-untranslated regions(due to selection of poly(A) sites nearer the 5’ end of the mRNA) is seen in rapidly growing cells (such as stem cells and cancer cells). The consequences of this seem to be significant, as it seems as if shorter RNAs are more abundant, owing to an absence of RNA sequences that destabilize the mRNA.  In other words:  shorter 3′ UTRs = higher expression = more growth/less regulation.

The means by which such global trends in poly(A) site choice may be accomplished are not clear; this will undoubtedly be one of the areas of interest in the field of polyadenylation over the next few years.

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