An RNA helicase scoots through the spliceosome to advance the process of mRNA splicing. And some other tricks.
In our cells, virtually all mRNAs transcribed from DNA have to go through an editing process to cut out intervening junk called introns. This process is called splicing, and its evolutionary origin, later elaborations, and current mechanism are all quite interesting. Life didn't start with introns, and only eukaryotes have them as a regular feature of their genomes. They appear to have arrived with the bacterium that became our mitochondria, which come from a lineage that has (relatively few of) what are called group II self-splicing introns. These are RNA segments that behave a bit like transposons, being able to jump into DNA, and then reverse-transcribe that segment into a copy of itself in genomic DNA.
The ur-eukaryote seems to have had an incredibly prolific infection, which left its host genome riddled with these bits of DNA. A key point is that, in group II introns, their splicing out of a transcribed RNA message is auto-catalytic- entirely mediated by their own RNA structure. They are self-propagating parasites, which have, over time in eukaryotes, been tamed to become fertile aspects of our own gene regulation and evolution. For example, introns often fall between protein domains, allowing these relatively compact modules of protein structure to be replicated, moved, and plugged, via rare mutational events, into new settings to contribute new functions to existing or novel proteins.
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| A map of a group II self-splicing intron. In red are the ends of the host RNA (or DNA) which are to be either jumped into or excised out of. The rest is the structure of the intron, which carries its own catalytic ability to do these reactions. This kind of thing is what appears to have turned into our own splicing and intron/exon systems, since the core catalytic mechanisms, such as the use of lariats and branch points and RNA catalysis, are the same. |
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| Representation of the core spliceosomal reactions in eukaryotes, which result in a free lariat form of the excised intron, and the joined exons, which go off to code for their protein. "SS" stands for splice site. The sequences in red characterize introns. |
The mechanism of intron excision in our cells is, at its core, still RNA-based, even though there are now also hundreds of proteins involved in the rather massive machinery of what is now called the spliceosome. It is clear that over evolutionary time, what was originally an unwelcome and shocking invasion of proto-mitochondrial introns into the proto-nuclear host genome has been regulated, speeded up, accessorized, and integrated into our normal method of gene expression. The spliceosome is the result- a huge and dynamic complex that uses key bits descended from the original RNA catalytic components to guide and catalyze the splicing reaction.
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| Representation of the core splicing reactions, with the key small RNAs added in (U1, U2, U4, U5, U6). These both guide (by direct RNA-RNA hybrid formation) and perform catalysis at the two chemical bond-breaking/reforming steps. |
There are three key locations seized upon by the spliceosome. First is the 5 prime splice site- the end of the coding exon and beginning of the intron, typically a "G" nucleoside at the start of the intron. Second is the branch point, an "A" near the end of the intron, which is where the chemistry of splicing begins. And third is the 3 prime splice site- the end of the intron, with another "G" nucleoside, next to the beginning of the next coding exon. While the first two sites are specifically recognized by RNA components of the spliceosome, (U1 at the 5 prime splice site and U2 at the branch site), the 3 prime splice site is simply recognized by scanning for the first "AG" downstream of the branch site.
The first reaction is to bring the branch site and the 5 prime splice site in proximity, such that the branch site A covalently invades the G at that site and displaces it, releasing the exon end and forming a loop (called a lariat, in red above) in the intronic RNA. The second reaction is to bring the 5 prime exon end over to the 3 prime exon end, and similarly prompt and invasion that links them, displacing the intron entirely.
So simple to describe, but not so easy to do. Accuracy is paramount, since the three-codon reading frame of mRNA would be destroyed by even a 1 nucleoside error in splicing. Splicing now gates the export of mRNA from the nucleus, so that only fully and accurately spliced transcripts get out to the ribosomes in the cytoplasm for translation to protein. This gating has been considered by some the very reason that the nucleus exists at all- a way to solve some of the knotty problems that arose in very early eukaryotic evolution when all these introns invaded.
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| Another reaction scheme of splicing, showing the key RNA and some other proteins along the way, principally the key helicases that help drive things forward. Note where PRP2/ATP comes into the picture, just as the complex is preparing for the first catalytic step. |
Be that as it may, it is clear that the originally RNA-only mechanism changed over time by accreting proteins that each decided they had something useful to add to the process. At the same time, the RNA got separated into several pieces (on independent genes) that could then be carried and precisely manipulated by these helping proteins. The spliceosome now involves five distinct small RNAs and over 200 proteins, which engage in a complex ballet of sequential steps. A special class of these proteins, the helicases, are the subject of a recent paper that provides new structural information. Helicases are proteins that can use the power of ATP to unwind DNA or RNA, or just chug along it. At least eight such proteins participate in splicing.
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| Structures from a recent paper, showing how PRP2 (at bottom, in violet) chugs its way along the mRNA intron (blue) into the very heart of the spliceosome complex, partially evicting the SF3B1 protein (green), among others, and prompting many other changes. At top is shown the 19 nucleoside stretch of the mRNA that was traversed, getting close to the branch site "A" in red. |
The paper makes the interesting observation that, structurally, most of the helicases reside on the periphery of the spliceosomal complexes, while the catalytic and guiding RNA are, naturally, at the center. They use a mutant form of one of them, Aquarius, to freeze spliceosomes in a key conformation just before the branch site and 5 prime splice sites are brought together. In combination with a bunch of other structural work by others in this and other labs, they show that one dynamic event is the tracking by a second helicase, PRP2 (violet, above), that brings it from its peripheral position (b, at bottom) along the intronic mRNA (blue strand) into the core of the splicesome near the U2 RNA (c; U2 is not shown here). They show that PRP2 traverses 19 nucleosides (top, a), a rather remarkable trip that forms part of the sequence of events that brings the branch site and 5 prime splice site close to each other.
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| Further structures, focusing on the catalytic site and RNAs. Note how the branch site (red, "BS-A") is, after the action of the Aquarius helicase, (third panel), brought in tightly close to the 5 prime splice site (green, "5'SS) in the C, or catalytic, complex. The U2 and U6 RNAs then have an easy job of bond-exchanging catalysis. |
So it turns out that these helicases appear sometimes to be used as ratchets, that start on the outside of the complex. Once activated by some prior trigger, they pull on a thread in a way that helps to overall process forward. The progression of PRP2 into the spliceosome core evicts a bunch of other proteins and activates the other helicase Aquarius. That protein is likewise positioned perpipherally but is hanging onto another thread of the intronic RNA and helps to further push the branch and 5 prime splice sites together, in a way that finally leads to the desired reaction. Note in the image above that it is the RNAs that occupy the central reaction site- the intron in blue (green), and the U6 and U2 RNAs, which catalyze this first key reaction of splicing.
RNAs are not great catalysts, so it is understandable that, as in the case of translation by the ribosome, a bunch of proteins shoehorned their way into the process (over evolutionary time) in ways that evidently made splicing more accurate and more rapid. Indeed, yeast cells get along without the Aquarius protein at all, though they otherwise have a very similar splicing apparatus, showing that the accretion of proteins on the spliceosome did not end in very early stages of eukaryotic evolution, but continued through the origin of metazoans, and may still be continuing. The added proteins did this through using their talents for precise spatial positioning, and for the use of energy (from ATP) to drive things ahead, if only by intricate conformational ballet rather than direct catalysis.
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