Late last fall, I published a short review in WIRES RNA that discussed some curious findings coming out of the growing community of plant scientists whose research touches on mRNA polyadenylation. When we think about the polyadenylation machinery, it is reflexive to consider that the core subunits (the CPSF, CstF, CFIm, and CFIIm subunits) should be essential. Indeed, this is the case in yeast and mammals, as far as one can tell. It is thus very surprising that Arabidopsis is able to grow (sometimes, with almost imperceptible phenotypes) in the absence of several supposedly core subunits. The list of dispensable proteins in plants includes CPSF30, FIP1, CstF77, and CstF64.
It’s been seven years or so since I updated this blog. In this post, I hope to summarize a direction my lab has taken in this time. If I am successful, you will see how interesting and exciting this research is, and the new directions we are exploring.
For the better part of 12 years, a main focus of research in my lab has been alternative polyadenylation in plants. I summarized a couple of seminal papers previously – here and here. One of the take-home points of this research was the scope of alternative polyadenylation – how many different poly(A) sites could be used, how usage shifts, and the impacts that shifts in poly(A) site choice have on gene expression. Since 2013, we have published many additional papers on alternative polyadenylation. The three summarized here help to develop a theme that guides current research in my lab.
By way of introducing this short entry: as is probably true for most blogs that discuss various and sundry aspects of science, I have tended to focus on reviews or peer-reviewed research papers – “the literature”. There is, however, a whole lot more to the lab than these finished and polished products. What I want to do with this entry is a bit different. Instead of talking about a complete study, I thought I would talk (briefly) about some results from my lab that, for various reasons, never found their way into print. Ideally, someone will read one of these essays and speak up, telling me just what is going on and how it fits in with other data or models.
The following is one such example, a result that is curious and perplexing. I chose it because it comes with pretty pictures, and because it is a segue for another essay that I will post in the future. The data is from a thesis of a student of mine – Kevin Forbes. The experiment itself is 7-10 years old (I have forgotten just when this study was done), and I made sure that Kevin would be OK with this before I posted anything.
The bi-annual Cold Spring Harbor Laboratory Meeting on Eukaryotic mRNA Processing is one that I try to attend on a regular basis. The last two meetings (2009 and this year) posed special problems for me, since I am also the driver and mule for Amy’s moving trip to Juniata College. The two institutions – CSHL and JC – don’t seem to “talk” to each other, and move-in has been coincident with the meeting (basically, 1 day apart, not enough time to drive to PA, return, and fly to NY). This means that I have ended up driving from Lexington to Cold Spring Harbor for the past two meetings. Load the car up with a dorm room, drive to Huntingdon PA, unload, and just continue to Long Island.
Well, it turns out that this was a pretty fortuitous choice of travel this year. The 2011 Eukaryotic mRNA Processing Meeting was, as usual, an exciting and productive one. But it may well be remembered as much for the bookends of the meeting – the eastern seaboard earthquake that ushered the meeting in on the 23rd, and Hurricane Irene, that necessitated some creative re-scheduling of the last day and a half of the meeting. Many participants were busier Friday re-scheduling shuttles and flights than listening to presentations. I was able to leave at the crack of dawn Saturday and beat the storm by about half a day.
The bookends aside, the meeting was excellent (as usual). I won’t post specifics here (CSHL has rules about commentary and disclosure that I will give a wide berth to). A few themes do merit mention. One is that polyadenylation and mRNA 3′ end formation was topical this year. This is due largely to studies such as I have discussed here and here. More and more labs have begun to look at alternative polyadenylation in the context of gene regulation and clinical outcomes, and the number of talks and posters that touched on polyadenylation was gratifyingly large.
A second theme was one that has been developing for the better part of a decade. It has become apparent that the various chapters in the life of an mRNA are not separated, either in time or space. The connections between the many steps – transcription initiation, elongation, termination, capping, splicing, polyadenylation, transport, translation, etc. – are being revealed in ever more fascinating detail. This was evident throughout the meeting.
A third theme was technical. In a nutshell, high-throughput DNA sequencing as applied to RNA has become all the rage. Lots of people are using variations on the themes I describe here and here to study alternative polyadenylation. (I hope to be able to discuss additional plant studies in the near future – stay tuned.) This in addition to other RNA-Seq applications, ChIP-Seq, CLIP-Seq, CRAC (see the brief mention near the bottom of this site), and other acronym-encoded approaches. (I’m kicking myself for missing an opportunity to come up with my own clever term. Oh well.) As sequencing becomes more affordable, I think that this trend will continue.
A short time ago, I mentioned this article. This study was the product of a collaboration between five laboratories – two plant poly(A) labs, a seed biology lab, and two bioinformatics groups. As the abstract indicated, this paper describes the results of a characterization of polyadenylation in plants using so-called Next Generation DNA sequencing technology; as such it is an addition to other recent studies, albeit the first (to my knowledge) that deals with plants.
I’m more than happy to answer questions about the paper in the comments. What I will do in the essay is described one of the more perplexing findings, and “amend” the PNAS paper with a few illustrations that we couldn’t include in the paper (even the online Supplemental Files – we maxed out the print and SI page limits).
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.
The RNA 2010 Meeting has come and gone. Previously, in a sort of preview of coming attractions, I gave a list (from the conference web site) of the many invited speakers. What I thought I would do here is toss out some random comments, to give readers a small taste of the meeting. (One aside – the abstracts are not “open access” and attendees are asked in the abstract book to not cite anything without authors’ consent. This means that I won’t be very explicit about the individual talks or posters. However, in a few instances, I will provide links to related papers.)
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.
Most genes in eukaryotes (well, at least eukaryotes that are not Saccharomyces cerevisiae) possess introns, sequences that are transcribed by RNA polymerase II and subsequently spliced out from the primary transcript. Introns have been the subject of tremendous interest since their discovery in the 1970’s, and have provided much insight (and grist for controversy) into subjects as disparate as junk DNA, the RNA World, and mechanisms of gene expression. Among the still-unresolved matters today has to do with the timing of splicing – is it cotranscriptional* or does it occur after polII has released the transcript.
The case for co-transcriptional splicing has been built in part through numerous studies that reveal physical connections between splicing factors and the transcriptional complex; many (most) of these involve the so-called CTD (C-Terminal Domain) of RNA polymerase II. (This recent review summarizes this emerging field.) The general idea is that, owing to the association of splicing factors with the CTD of polII, they are able to bind the nascent transcript and initiate splicing before polII has completed the synthesis of the primary transcript.
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.