Safe at home!

Nothing like a steal of home to get a crowd going.

Of course, what is missing from all the buzz about this is how Ellsbury set up the first run of the game with his base-running, coaxing a throwing error out of the Yankee’s third baseman.

These ain’t your grandpa’s Red Sox.

Convergent transcription, polyadenylation, RNA editing, and regulation

One of the mechanisms by which polyadenylation may contribute to the regulation of gene expression (on paper, at least) involves gene pairs that are situated near each other and transcribed convergently.  In these instances, polyadenylation and transcription termination need to occur to prevent the production of RNAs that are anti-sense to the two members of the convergently-transcribed gene pair.  Overlapping transcripts could lead to the formation of double-stranded RNAs that could in turn trigger regulatory mechanisms, resulting in altered accumulation of the corresponding transcripts.

It is in this vein that a recent study from Gordon Carmichael’s lab at the University of Connecticut is of interest.  Briefly, these authors report that the early-to-late transition in gene expression in cells infected with the mouse polyoma virus is accomplished (at least in part) by a reduction in polyadenylation efficiency of the primary transcript encoding the so-called late genes.  Interestingly enough, this reduction in polyadenylation efficiency seems to be due to A-to-I editing of the region around the polyadenylation signal.  This editing in turn may be traced to an overlap of the early and late transcripts, such that double-stranded RNAs (the substrate for the A-to-I editing complex) that include the late polyadenylation signal are produced and edited before pre-mRNA processing occurs.

SOC1 and the RNA Underworld

Earlier, I described studies of the so-called SOC1 and FUL genes of Arabidopsis, genes that when mutated in concert change the growth habit of the plant is most remarkable ways.  A report that just came up on Plant Cell Online links one of these genes with one of the mechanisms by which RNAs are turned over in the cell.  Briefly, this study reveals that SOC1 expression is subject to posttranscriptional control, and that this control is linked with a component of the machinery that mediates nonsense-mediated decay (NMD) in plants.  This finding may be of interest for a number of reasons.  One is that NMD hasn’t yet been linked with lots of regulation in plants – it occurs, and we may infer conceptual links between alternative RNA processing and NMD, but much remains to be learned.  A second is that SOC1 functioning, previously implicated in important macroevolutionary transitions in plants, may be altered by many evolutionary processes, including those that affect RNA levels through NMD.

The abstract:

SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) is regulated by a complex transcriptional regulatory network that allows for the integration of multiple floral regulatory inputs from photoperiods, gibberellin, and FLOWERING LOCUS C. However, the posttranscriptional regulation of SOC1 has not been explored. Here, we report that EARLY FLOWERING9 (ELF9), an Arabidopsis thaliana RNA binding protein, directly targets the SOC1 transcript and reduces SOC1 mRNA levels, possibly through a nonsense-mediated mRNA decay (NMD) mechanism, which leads to the degradation of abnormal transcripts with premature translation termination codons (PTCs). The fully spliced SOC1 transcript is upregulated in elf9 mutants as well as in mutants of NMD core components. Furthermore, a partially spliced SOC1 transcript containing a PTC is upregulated more significantly than the fully spliced transcript in elf9 in an ecotype-dependent manner. A Myc-tagged ELF9 protein (MycELF9) directly binds to the partially spliced SOC1 transcript. Previously known NMD target transcripts of Arabidopsis are also upregulated in elf9 and recognized directly by MycELF9. SOC1 transcript levels are also increased by the inhibition of translational activity of the ribosome. Thus, the SOC1 transcript is one of the direct targets of ELF9, which appears to be involved in NMD-dependent mRNA quality control in Arabidopsis.

The citation (hopefully, I will remember to update it once the paper comes out in print with the updated link for the paper copy):

Hae-Ryong Song, Ju-Dong Song, Jung-Nam Cho, Richard M. Amasino, Bosl Noh,  and Yoo-Sun Noh. The RNA Binding Protein ELF9 Directly Reduces SUPPRESSOR OF OVEREXPRESSION OF CO1 Transcript Levels in Arabidopsis, Possibly via Nonsense-Mediated mRNA Decay.  Plant Cell Advance Online Publication, Published on April 17, 2009; 10.1105/tpc.108.064774

Pretty incredible

That’s a subdued way to described last night’s NCAA Division I hockey championship game between the upstart Miami (OH) RedHawks and the Boston University Terriers.  The underdog RedHawks dominated, absolutely dominated the game, especially the first 19 minutes of the third period.  With 60 seconds to go, they held a 3-1 lead.  It looked like the school would win their first national championship in any sport.

But then …!!!  BU had been playing without a goalie since about the 3 min mark (well, except when there were face-offs outside of the MU zone).  The extra skater payed off when they scored with 59 seconds left in the game, and again with 17 seconds remaining.  Stunning, especially since they had been lethargic and totally outplayed until this point.

This set up overtime.  Sudden death overtime in hockey is the most exciting, tense, enthralling spectacle in sport (IMO, at least).  In this case, it ended 11+ minutes into OT, a slapshot deflecting off a sliding MU defender over the goalie’s shoulder ….

Read more here and here (Dan Shaughnessy already calls it the greatest college hockey game ever).  I haven’t seen that many, and I expect RedHawk fans would call it more a nightmare, but it was pretty exciting.

Rooting interests – I’m a Bostonian at heart, but my good friend and collaborator is a professor at Miami.  So I’m pretty torn by this.  In a good sort of way. (Well, except in knowing that my alma mater, UMass Lowell, lost their chance to get into the NCAA tournament by one goal to, um, BU.)

One more dig – the Frozen Four beat the heck out of the Final Four this year.

Targeted gene insertion in plants

One of the strategies used by scientists to introduce foreign genes into living cells involves the targeted insertion of the foreign gene into a pre-determined chromosomal position.  This approach has been used to great effect in (for example) yeast and transgenic mice to disrupt genes (hence the term “knock-out mouse”), and thus study their functions.  Except in chloroplasts, similar strategies have been hard to perfect in plants.  However, new technologies may be bringing us closer to the day when targeted gene disruption is an accessible methodology in plant systems.  A recent report from Joe Petolino’s group at Dow Agrosciences provides an example.

Briefly, this group is developing tools that capitalize on the ability to design tailor-made DNA binding proteins that recognize specific sequences.  Over the past several years, much progress has been made in understanding and adapting the DNA binding properties of a specific class of proteins known as zinc-finger proteins.  It turns out that the chemical rules for specificity in some classes of these proteins can be delineated, and that different sequence specificities can be engineered in a rational way by linking a desired sequence of bases with amino acid side chains predicted to facilitate interactions with the base.  It also turns out that one can target double-stranded DNA cleavage to specific sequences by linking endonucleases with sequence-specific DNA-binding proteins. Putting these two things together – tailor-made DNA binding specificity and targeted double-stranded cleavage – brings us technology to introduce double-stranded breaks at pre-determined chromosomal positions.  (These chimeric proteins are known as zinc finger nucleases.)  Add to this the realization that transgene integration occurs at double-stranded breaks, and one gets to the point described in the recent paper from Dow Agrosciences.

The abstract from the paper is given beneath the fold.  What the authors did was design zinc-finger proteins that would recognize sequences in any of a number of exogenous reporters of endogenous host genes and use a battery of physiological and PCR-based assays to evaluate the efficacy of their strategies.  Thus, they showed that they could promote intra-genetic recombination so as to restore a split reporter gene (encoding green fluorescent protein, or GFP).  They then showed that they could promote intergenic recombination between two  DNA molecules introduced transiently into tobacco cells using Agrobacterium; in this instance, they assayed for the reconstitution of a transgene that confers resistance to a herbicide (Bialaphos).  Finally, they demonstrated targeted insertion of foreign DNA into a tobacco chitinase gene.  This demonstration involved the attempted insertion of a Bialaphos gene into the genome; when used in conjunction with the tailor-made zinc finger constructs, as many as 10% of the recovered Bialaphos-resistance cells has the desired insertion.

While 1 in 10 may not seem to be very efficient, it far exceeds random T-DNA insertion, and brings the possibility of targeted gene disruption within the realm of possibility in plants.  Beyond the utility of this technology in the lab, it will also provide crop scientists with ways to genetically modify plants such that unanticipated molecular consequences (inadvertent inactivation of endogenous genes by random insertion events, disruption of genetic circuits due to triggering of RNAi, to name two) are avoided.