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Splicing outcomes: targeting for exon skipping or intron inclusion

Someone asked me why some Morpholinos usually cause exon skips and others cause intron inclusions...

U1 and U2 (or U11 and U12) snRNPs mark the positions on pre-mRNA of the splice junctions for the spliceosome. There is a U1 snRNP that binds in the intron near the e2i2 junction and a U2 snRNP that binds on the other side of the intron at the i2e3 junction.

Figure 1
Figure showing normal RNA splicing.

Morpholinos targeting splicing are usually targeted to block the binding sites of these snRNPs. Consider the splice junctions of the second intron. If you block the U1 snRNP binding site with an e2i2 oligo, the spliceosome will match the U2 snRNP at i2e3with the next available U1 snRNP upstream, which is bound at the e1i1 junction. This splices the e1i1 junction to the i2e3 junction, eliminating i1, e2 and i2.

Figure 2
Figure showing exon 2 skipping.

Now consider what happens if you block the e1i1 junction with a Morpholino. The U1 can't bind to its e1i1 site, but the U2 at the i1e2 site has no other upstream U1 it can be redirected toward to make a splice. In this case, the usual result is for no splicing to occur at i1; that is, you see an i1 inclusion in the mature mRNA.

Figure 3
Figure showing intron 1 inclusion.

Because the very first (e1i1) and very last splice sites have no splice sites past them to which splicing can be redirected, blocking them causes a failure to splice (intron inclusion). Blocking any of the internal splice boundaries (i1e2, e2i2, i2e3, etc.) usually causes the adjacent exon to be eliminated from the mature mRNA (or, in the common jargon, causes the exon to be skipped). Here is an illustration of intron inclusion resulting from blocking the last splice junction.

Figure 4
Figure showing intron 3 inclusion.

Other things can happen. Activation of cryptic splice sites can cause partial exon excision or partial intron inclusion. Sometimes an oligo causes a double exon skip. Another possible outcome is a failure to splice of an internal intron, causing its inclusion in the mature mRNA. However, the most common outcomes are a intron inclusion for the first and last splice sites and an exon excision (skip) for all other splice junctions.

Figure 5
Figure showing possible splice outcomes.

An entirely different approach to modulating splicing is to block the binding sites of splice regulatory proteins (splice enhancers, splice suppressors). I'm mentioning it here, but not discussing it more.

Typically splice-modifying oligo efficacy is tested with reverse-transcript PCR followed by gel electrophoresis of the PCR product. It can take several primer pairs to determine what happened. Sometimes putting a primer in each of the exons adjacent to the target can reveal a clean excision of the targeted exon. To detect the exon 2 skip in the second figure above, you would typically put a primer in exon 1 and another in exon 3. Other common approaches are to move a primer an additional exon away to look for a double-exon-skip or to place a primer in intronic sequence to detect an intron inclusion. For cryptic splice site activation where the cryptic site is close to the wild-spliced site, sequencing the PCR product can reveal small changes. When choosing primers, be sure that you plan to make RT-PCR products of a hundred bases or so when the exon is missing; that way the RT-PCR product is long enough it can take up plenty of an intercalating fluorescent dye and be readily visible on the gel.

If you do not see an obvious single-exon-skip by looking at the RT-PCR product on an electrophoretic gel, one of these alternative splicing outcomes might be the reason (but the most likely outcome is the single-exon skip). Load the gel lightly and watch for dimming of the wild-spliced band in the MO-treated vs. control lane (normalized against a housekeeping gene band intensity). If the band is over-saturated with too much RNA , it can be harder to see the dimming of the band if some of the transcript is diverted to a different splicing fate, but if you have a lightly-loaded gel then it should be obvious if you lose some band intensity (normalized against a housekeeping gene). If nonsense-mediated decay is occurring, you might not see much or any mass-shifted band from splice-modification, but you should see the wild-spliced band become more dim.

One more time but drawn a bit differently - this is why blocking an internal splice site usually causes exon excision, but blocking the first or last splice site usually causes intron inclusion.
Figure 6
Figure comparing expected splice outcomes.

Comments

Dear Professor : Re. The above mentioned paper please allow me a fundamental questiom : From my point of view as structural design engineer I see the splicing fact as an engineering problem .....assume that there is a gene with ten exons 1 to 10 , then assume that in some tissue , state and time an specific isoform is needed : exon 10 - exon 7 - exon 2 then the question is : how the spliceosome recognize and detect and and select and connect 10 -7 - 2 exons while there are 720 combinations of three from ten exons available ?? I mean what is the multiplex code that is : If X then n10- n7- n2 exons ?? Where X is the cell state in time tn and n are the number of exons in the gene ....Gow the spliceosome knows that X equals 10-7-2 in particular ???? I appreciate very much getting your response . Thanks and best regards Nady Shamy with respect

Hi Nady, good question. Some of the exons undergo alternative splicing, while others seem to be in every RNA produced form the gene. So, we can ignore the invarient exons and look at the alternatively spliced ones. Of these, many are regulated by splicing proteins that bind to the RNA and signal to the spliceosome whether or not a particular exon should be spliced out. These splice-regulatory proteins are designated by where they bind and what they do, for instances: exonic splice enhancers (ESE), intronic splice suppressors (ISS) etc. Morpholino oligos are sometimes used to bind to RNA at the recognition site of a splice-regulatory protein, preventing it from binding and influencing splicing. There are other splicing effects of different mechanisms, such as when the splicing-out of one exon is correlated with the splicing-out of an adjacent exon without apparent regulatory protein involvement with the adjacent exon. Keep in mind that these are evolved systems and so unusual networks can appear. Everything is fluid though mutation; for example, an invarient exon can have a splice-regulatory binding site created though mutation which then causes the exon's removal or the end of an exon can be redesignated within an intron be mutational creation of a new splice site, lengthening the exon. By the way, I'm not a Professor but instead work in industry.

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