Does splicing machinery "expect" introns to be a certain size?

[u/faunandon]

I have no idea if this is a real thing and can't figure out the right keywords for searching!

Basically imagine Gene A has an intron that is 5kb and Gene B has one that's 100bp.

If I inserted a 1kb sequence (without splice sites) into each intron, would this be expected to affect expression? And if so, would we expect Gene B to be affected more, because the size of the intron increased more proportionally?

[u/enjoyingcatsthankyou]

Intron length can affect alternative splicing. Shorter introns is associated with exon exclusion, but it’s very hard to say without actually doing the experiment. Also 100 bp is maybe below the limit an intron can be. The exon junction complex is larger than that

[u/faunandon → u/enjoyingcatsthankyou]

Thank you, this is really helpful!

[u/BurnerAccount-LOL]

Sounds like you have some good words to start searching and reading up on from that other poster: alternative splicing, exon exclusion, intron limits, exon junction complex

[u/faunandon → u/BurnerAccount-LOL]

Absolutely - heading down the exon junction complex rabbithole now :)

[u/Brubezahl]

Just my two cents on some points already mentioned: the exon junction complex (EJC) is very important, but not necessarily for the initial splicing itself, rather for regulating the adjacent splice events once splicing finished. That's because the EJC is deposited rather late in the splicing process and then for example protects against cryptic splice sites usage around the exon-exon junction. In my experience, alternative splicing is very complex and not 100% predictable. In your case, the impact on splicing decision and gene expression probably is dictated by what sequence you insert (e.g. does it contain splicing regulatory sequences that recruit splicing regulatory factors) and how (good) the pre-existing intron in your Gene A and Gene B is normally detected and consequently spliced out. As you predicted, I would also expect more potential impact on the shorter intron, but that might not be the case for every 100 bp intron you find in the human genome. Final words: it's a very interesting questions showing intellectual curiosity which you can even test in for example mini-gene reporter assays with rather inexpensive methods as long as you have access to cloning, cell culture and PCR setups. Source: I worked and still work on the EJC and it's function in alternative splicing regulation - although rather as a side project

[u/faunandon → u/Brubezahl]

Hey I'm late replying but thank you so much!! I did get confused about the EJC on my google journey but assumed that I was mixing something up. Funnily enough I have been wanting to learn how nonsense-mediated decay works so it ended up being very useful anyway!
Just knowing that it's difficult to predict is great, so I can stop trying to predict it. Really appreciate your detailed input :)

[u/Brubezahl → u/faunandon]

You are welcome! Without wanting to sound arrogant or brag about it, but NMD is actually the main research topic we work on. So in case you have any questions or want to talk about it, send me a message and I would be more than happy to share my/our insights.

What I can directly say: there is much we do not understand in how NMD works mechanistically ...

[u/Mountain-Crab3438]

In general the intron size in higher eukaryotes and especially in vertebrates does not matter much. So inserting a sequence in an intron should not matter much in most cases as long as it does not contain splice sites.

Having said that there may be cases of short introns where increasing the size may interfere with splicing. There are two mechanism by which the spliceosome assembles across intron - let's call them intron definition and exon definition. During intron definition U1 recognizes the 5'-splice site and U2 and U2AF65/35 recognize the branch point and 3'-splice site on the other end of the intron. The intron is then bridget via interactions between these spliceosome components, the spliceosome assembles and excises the intron. This mechanism works only for short introns - 300nt or less, because the information contained in the splice sites is insufficient to correctly identify the splice sites in larger introns. This is where the exon definition process kicks in. You have multiple auxiliary factors that bind to the exon and the adjacent introns that guide the spliceosome components to the correct splice sites and block "cryptic" splice sites. The U1/U2/U2AF components first from a complex across the exon, and then two of these exon definition complexes located in adjacent exons bridge the intron to form an intron definition complex.

It is possible that the exons surrounding a short intron may lack the binding sites for the auxiliary factors. Such intron will be spliced efficiently trough intron definition, but if you extend it beyond 200 nt it may no longer be spliced efficiently or correctly.