RNA is everywhere …

This essay is  follow-up to a previous entry, the second in what may become a series of unpublished but very real results.

For those of you who have downloaded and read Kevin’s thesis, you will have noticed that much effort was expended in characterizing an unusual inhibitor of poly(A) polymerase.  Kevin started in my lab in the days before the Arabidopsis genome was completed; in those days, my lab was committed to pursuing biochemical approaches to understand polyadenylation.  Unfortunately, it was not possible to observed authentic processing and polyadenylation activity in a plant-derived nuclear extract.  (The reasons for this remain unknown, to this day.  Quinn Li’s group has succeeded in detecting processing in an Arabidopsis extract, but even in this case the processed RNAs are not polyadenylated by endogenous poly(A) polymerase.)   However, it had been reported, in the mammalian and yeast literature, that one or more polyadenylation factor subcomplexes could inhibit the non-specific activity of poly(A) polymerase.  The latter is an enzyme that can be detected, assayed, and purified from plant sources.  Thus, it made sense to use the inhibition of this activity as a sort of assay for other polyadenylation factors.

This was where Kevin started.  Sure enough, he was able to identify a very effective inhibitor of poly(A) polymerase.  After ruling out trivial explanations (it wasn’t a nuclease or protease), he pressed on to purify the inhibitor, with the goal of obtaining DNA clones that encoded the inhibitor (or its subunits).  One of the components that copurified with the inhibitor was an interesting variant of histone H1.

In addition to showing that the inhibitor was not a nuclease or protease, Kevin tested the efefcts of proteases and nucleases on the activity of the inhibitor.  Not suprisingly, it was sensitive to protease treatment; this showed that it is a protein.  However, the inhibitor was also sensitive to nuclease treatment, suggesting a role for RNA in its activity.  Kevin isolated nucleic acids from the purified enzyme (in those days, this meant phenol extraction and ethanol precipitation), end-labelled anything that came out, and analyze things on sequencing gels.  Surprisingly, what he found was a population of one or more small RNA species.  The figure from his thesis:

(The inhibitor was called PPF-B.)  Because U1 snRNP was known at the time to inhibit poly(A) polymerase, Kevin also did some northerns that showed that the inhibitor was devoid of U1 snRNA.  The unknown RNA in the PPF-B preparation was clearly larger than the stable RNAs seen in nuclear extracts (“extract” in the figure), and to this date has not been identified.

There’s more to this story in his thesis.  What remains curious (and, as is the case with the connection with H1, incites the overactive imagination) is the association of a small RNA (but NOT an siRNA or miRNA, since it is far too large) with poly(A) polymerase inhibitory activity.  Kevin did not follow-up on this, since the “end of the tunnel” for this project seemed years away.  So he instead turned his attention to the Arabidopsis ortholog of Fip1p, an effort that resulted in a nice thesis chapter and an interesting JBC paper.  The large RNA associated with the inhibitor still lurks in the back of my mind, but so far no obvious explanation has jumped out of the literature.  Maybe someone reading this will be inspired to ….

Cleveland Rocks!

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.

Plants are people too!

The first sentence from a recent paper in RNA:

Noncoding RNAs (ncRNAs) are widespread transcripts occurring from yeast to human, but their functions remain unclear (Kapranov et al. 2002; Rinn et al. 2003; Yelin et al. 2003; Cheng et al. 2005; Davis and Ares 2006;Neil et al. 2009).

Um, what the heck! ncRNAs have been known in plants for quite some time – at least 5 years.

Oh well.  The paper itself is interesting – talking about how short intragenic RNAs* modulate transcription of their associated gene.  The suggestion (based on experimental data) that the specific ncRNAs in this case are not transcribed by polII adds yet one more twist to the interconnections between polymerases, chromatin modifications, and regulation.

And, yes, it’s almost for certain that these things happen in plants.

The abstract:

Inter- and intragenic noncoding transcription is widespread in eukaryotic genomes; however, the purpose of these types of transcription is still poorly understood. Here, we show that intragenic sense-oriented transcription within the budding yeast ASP3 coding region regulates a constitutively and immediately accessible promoter for the transcription of full-length ASP3. Expression of this short intragenic transcript is independent of GATA transcription factors, which are essential for the activation of full-length ASP3, and independent of RNA polymerase II (RNAPII). Furthermore, we found that an intragenic control element is required for the expression of this noncoding RNA (ncRNA). Continuous expression of the short ncRNA maintains a high level of trimethylation of histone H3 at lysine 4 (H3K4me3) at the ASP3 promoter and makes this region more accessible for RNAPII to transcribe the full length ASP3. Our results show for the first time that intragenic noncoding transcription promotes gene expression.

The citation and link:

Huang YC, Chen HT, Teng SC. 2010. Intragenic transcription of a noncoding RNA modulates expression of ASP3 in budding yeast. RNA 16, 2086-2093.

* – short intragenic RNAs are non-coding RNAs (ncRNAs) that are transcribed from within a larger gene or transcription unit.  The ones described in this report are in the sense orientation, but antisense short intragenic ncRNAs are also possible.

Recap

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.)

“Where Did All the Flowers Come From?”

Carl Zimmer has a good article in the NY Times entitled “Where Did All the Flowers Come From?”   The article summarizes lots of interesting stuff, but I find the speculation regarding the evolution of the endosperm to be particularly though-provoking.  Of course, anytime one mentions genome duplication to me, visions of gene silencing and small RNAs begin dancing in my mind.  A recent article from David Baulcombe’s group merits mention in this context.  This paper describes a developmental study of RNA polymerase IV-derived small interfering RNAs (siRNAs).  The remarkable finding in this paper is the observation that the synthesis of many polIV-derived siRNAs is initated at the onset of the development of the maternal gametophyte, and that these siRNAs are in turn derived from the maternal genome(s) in the endosperm.  This has ramifications for the expression of the different genomes in the endosperm, for genome imprinting, and likely for the evolution of flowers and seed development in plants.

The abstract from the paper:

“Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci^{1, 2, 3, 4, 5}. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II^{2, 3, 6}. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.”

The citation:

Mosher RA, Melynk CW, Kelly KA, Dunn RM, Studholme DJ, Baulcombe DC. 2009.  Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis. Nature 460, 283-286 (9 July 2009) | doi:10.1038/nature08084.

More strangeness …

Awhile ago, I discussed a flurry of papers in Science that showed some curious aspects of transcription and promoters.  It seems as if every passing day brings a new report that pertains to the phenomenon.  A recent issue of Nature brings us two papers, back to back, that are relevant.  The bottom line is that bidirectional transcription is a widespread phenomenon, at least in yeast.  Moreover, this phenomenon is responsible, not just for divergent transcription of mRNA-encoding genes, but also for the production of so-called Cryptic Unstable Transcripts and other uncharacterized RNAs.  The abstracts and some brief commentary are beneath the fold.

RNA and kitchen tools – Slicers, Dicers, and CRISPRs

RNA-based regulation is all the rage in biology today.  The more familiar mechanisms involve small RNAs such as microRNAs and silencing-associated RNAs.  The biogenesis and functioning of these RNAs involves enzymes and complexes that have been termed, among other things, Dicers and Slicers.  These subcellular kitchen utensils work by processing either the small RNA precursor or the base-paired target RNA.  This mode of regulation is most often associated with eukaryotes, and indeed homologous enzymes and mechanisms are not found in prokaryotes. However, systems with remarkable functional similarity may occur in bacteria.  A recent review by Sorek et al. brings one such example into focus.

One curious feature of bacterial genome is the occurrence of arrays of direct repeats in which the repeated units are separated by so-called spacers of unique sequence unrelated to the repeat units.  The sizes of the repeat units vary from bacteria to bacteria, ranging from between 24 to 47 bp.  Likewise, the spacer sizes vary from 26-72 bp.  These arrays are flanked by an apparent leader sequence, and yet again by arrays of protein-coding (CAS) genes, the number and composition of which vary considerably from bacteria to bacteria.  The general arrangement is shown in the following figure, which is part a of Figure 1 from Sorek et al. (shown beneath the fold):

Strange things at promoters

A group of interesting papers popped up on ScienceExpress this past week.  These papers (by Core et al., Seila et al., He et al., and Preker et al.) all describe characterizations of unusual patterns of transcription in human cells.  The bottom line (well, one bottom line – there are lots of interesting data in these studies, and the nuances may take readers in slightly different directions) is that, for numerous promoters, transcription extends in both directions, not just in the one direction that is usually associated with productive (= leading to synthesis of a processed and translated mRNA) transcription.  Moreover, this bidirectional transcription is quite distinct from that associated productive transcription, in that it yields short and relatively unstable RNAs.  More elaboration follows below the fold.  As always, enjoy.

The Small RNA World – a brief introduction

The first reflex when coming across the title of this blog is, most likely, that it is a blog that mentions microRNAs and small RNAs. Up until now, I suppose that I’ve been a disappointment, as the scientific focus has been on the subject matter of my lab – polyadenylation. But this changes with this essay, an overview of the field of small RNAs. My goal with this overview is to lay a foundation to which I can refer in other contexts. As always, enjoy (and feel free to ask questions or correct any mistakes you find).

The 2008 Winners

… of the Albert Lasker Basic Medical Research Award are Victor Ambros, David Baulcombe, and Gary Ruvkun.  These scientists are pioneers in the field of small RNAs, and have helped dissect the process in animals and plants.  Some snippets from The Lasker Foundation announcement:

The 2008 Albert Lasker Award for Basic Medical Research honors three scientists who discovered an unanticipated world of tiny RNAs that regulate gene activity in plants and animals. Victor R. Ambros (University of Massachusetts Medical School, Worcester) and Gary B. Ruvkun (Massachusetts General Hospital, Boston, Harvard Medical School) unearthed the first example of this type of molecule in animals and demonstrated how the RNAs turn off genes whose activities are crucial for development. David C. Baulcombe (University of Cambridge) established that small RNAs silence genes in plants as well, thus catalyzing discoveries of many such RNAs in a wide range of living things. His findings led to the identification of the biochemical machinery that unifies numerous processes by which small RNAs govern gene activity.

Ambros, Baulcombe, and Ruvkun did not set out to unveil small regulatory RNAs. Ambros and Ruvkun were studying how the worm Caenorhabditis elegans develops from a newly hatched larva into an adult. Baulcombe, in a seemingly unrelated line of inquiry, was probing how plants defend themselves against viruses. All three investigators possessed the open mindedness, wisdom, and experimental finesse to entertain the possibility—and then verify—that tiny RNAs could perform momentous feats. Their work has led to the realization that these molecules are pivotal regulators of normal physiology as well as disease.

A few paragraphs later:

Across the Atlantic, David Baulcombe, then of the Sainsbury Laboratory in Norwich, UK, was studying how plants resist viruses. When he and others added to viral-infected plants unusual versions of viral genes, the mRNA copies of the normal genes as well as the newly introduced ones disappeared. Similarly, experimentally added non-viral genes suppressed activity of plant genes that contained similar sequences. Baulcombe proposed that such gene silencing occurs when RNAs embrace target mRNA—through typical Watson-Crick base-pairing—and promote destruction of the mRNA or interfere with its translation into protein. However, no one could find such RNAs.

Baulcombe reasoned that the predicted RNAs might have eluded researchers because the molecules were shorter than anyone imagined and thus, experiments had not been designed to detect them. In 1999, he and a postdoctoral fellow in his laboratory, Andrew Hamilton, devised a hunt specifically for small RNAs. They added test genes to plants and found 25-nt long RNAs that matched; furthermore, these small RNAs appeared only under conditions in which target mRNA activity was shut off. The stunning similarity in size between the plant and worm RNAs suggested that small regulatory RNAs exist in many organisms. Furthermore, it hinted at the presence of cellular machinery that dedicates itself to creating these precisely sized molecules and then uses them to quash gene activity.

Readers are encouraged to read the paper by Hamilton and Baulcombe that started to reveal the true scope of the RNA Underworld.  And another paper from Baulcombe’s group that ties in an underlying theme of this blog to the subject of small RNAs and silencing.  As always, enjoy.