Intron-mediated enhancement of gene expression

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.

On the functional significance of alternative splicing

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.

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.

Interesting stuff

While my yard is recovering from the ice, and I from today’s UK game, I thought I would toss out a few interesting abstracts that touch on important and contentious issues.  Peek beneath the fold and, as always, enjoy.

Cytoplasmic polyadenylation

The polyadenylation of mRNAs is usually thought of as a process that occurs in the nucleus, and indeed this is the cellular compartment in which pre-mRNA processing and polyadenylation does occur. However, mRNA polyadenylation is not restricted to the nucleus. Indeed, one of the more fascinating and important mechanisms that control gene expression during oogenesis and early development, stages in some organisms (such as animals) when the nucleus is not “active”, is mRNA polyadenylation. In these cases, the process occurs in the cytoplasm.

During oocyte development, a large population of maternally-encoded mRNAs are synthesized and stored for “use” in particular stages of development. These maternal mRNAs typically possess short poly(A) tails (20-40 nts) and are not available for translation (being “masked” by a complex of RNA-binding proteins). During oocyte maturation or following fertilization, these masked mRNAs become polyadenylated and thus activated for translation. This activation is a regulated process that helps to coordinate the ballet of gene expression attendant with meiotic maturation and early development. As such, it touches on many tangential phenomena (such as movement of stored mRNAs within the cell).

What is of particular interest for this blog is the nature of the mechanism that mediates polyadenylation in the cytoplasm. As indicated in the following figure, this mechanism includes some familiar players as well as some equally-intriguing partners.

The nuclease aisle

One of the themes that keeps popping up here is that of nucleases.  I thought I would post an adaptation of a table from a recent review by Ciarán Condon that lists the various ribonucleases in E. coli and B. subtilis.  The point is to illustrate the variety of nucleases that exist in bacteria, and to get readers to think more of the importance of RNA processing and, moreso, RNA turnover.

This table is adapted from Condon C (2007), Maturation and degradation of RNA in bacteria, Current Opinion in Microbiology 10: 271–278.  Enjoy.

The polyadenylation complex – Endonucleases

Messenger RNA 3’ end formation involves an RNA processing step.  It is thus natural to assume that the polyadenylation complex (as illustrated here) includes at least one subunit that is an endonuclease (an enzyme that cuts the RNA within the polynucleotide chain, as opposed to at the 5′ or 3′ ends).  Indeed, the complex does include such a nuclease.  However, just as there seem to be more RNA binding proteins than known cis element, there may be a multiplicity of endonucleases in the complex.  Specifically, at least two CPSF subunits (CPSF73 and CPSF30) have been shown to possess endonucleolytic activity in vitro (1-4).  The research community would probably consider that CPSF73 is the most probable candidate for THE processing endonuclease, but the possibility that CPSF30 serves this role, along with or instead of CPSF73, cannot be ruled out.  (Except perhaps in yeast, whose CPSF30 counterpart, Yth1, apparently lacks the nuclease activity seen in other eukaryotic homologs [5].)

Alternative polyadenylation

Polyadenylation is a process that pertains to the expression of almost all genes in eukaryotes.  In addition, as is the case with virtually every step in the process, polyadenylation is a step at which regulation may occur.  The most obvious way by which this might occur is via what is commonly known as alternative polyadenylation.  Alternative polyadenylation, in a nutshell, involves the use of alternative poly(A) sites within a single gene.  The general idea is shown in Figure 1 – any gene that has more than one poly(A) site has the potential to be processed at any of the various sites.

Regulatory evolution, agriculture, and RNA – a possibly interesting intersection

Much of the interest and excitement in the field of “Evo-Devo” today centers on the roles that changes in gene regulation may play in the evolution.  This mechanism (altering when and/or where a particular gene is expressed during development) stands apart from that concerning changes in the actual structure and function of individual proteins.  A recent study from Steve Tanksley’s lab brings this phenomenon into focus for this blog, and may tie together some different themes.

This study (Cong et al., Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication, Nature Genetics 40, 800-804, 2008 ) deals with one of the two processes (cell cycle control and organ number determination) associated with the enlargement of tomato fruit size in the course of domestication of this crop.  Many years of QTL mapping have led researchers to regions of the tomato genome involved in these processes.  The study by Cong et al. describes the end result of the characterization of one of these QTLs, a locus that is a major contributor to organ number (specifically, carpel number).