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Insertion in intron

How can I explain an insertion of 158 bp in intronic region suppress the gene expression??

I have one resistant and one susceptible line, I sequenced a gene in both and found 158bp insertion in intronic region in the susceptible line, where the gene expression is significantly lower compared to resistant line. How can I explain this?

For note: I have sequenced 1kb promoter region upstream of ATG but wouldn't see any sequence varaition

Thanking you!

sequence
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updated by Biostar Biostar • written by dadhokane • dadhokane

2 answers

It's entirely possible to reduce/abrogate splicing or change the splice acceptor site (e.g., by altering the polypyrimidine tract or lariat branch site), which typically results in out-of-frame transcripts that are subject to nonsense-mediated decay (NMD).

Point of clarification: by CDS sequencing, do you mean genomic DNA or RNA-derived cDNA? The former cannot not detect splicing errors or NMD, and even RNA-Seq may not be sufficiently sensitive for aberrant splicing (and NMD manifests as reduced expression).

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written by harold.smith.tarheel harold.smith.tarheel

I sequenced cDNA using Sanger sequencing

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written by dadhokane • dadhokane

Since it's a heterozygous variant, could you see both alleles when sequencing the cDNA?

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written by WouterDeCoster WouterDeCoster

No but just I have one allele sequenced, I can see the difference on gel

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written by dadhokane • dadhokane

So the allele carrying the insertion is not expressed?

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written by WouterDeCoster WouterDeCoster

???

I'm confused; are you saying that the cDNA is a different size by gel electrophoresis? Then how can the cDNA sequence be identical to the wild-type?

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written by harold.smith.tarheel harold.smith.tarheel

I agree it's not clear, likely a terminology problem, that's why I more explicitly reformatted my question.

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written by WouterDeCoster WouterDeCoster

I guess it could be a couple things. Like the RBS has moved and the reading frame is destroyed. But that would lead to a total knock out of the protein. It could be causing a splicing error can be giving you a truncated or mutated protein that is still being made but less effective. If you have any RNAseq data you could use that as well to find more evidence for this insert causing your mutation.

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written by skbrimer skbrimer

Splicing would be my primary suspect.

@OP: is this insertion homozygous or heterozygous? Which organism?

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written by WouterDeCoster WouterDeCoster

But no difference when I sequenced CDS in both, its exactly the same.

Its in wheat, its heterozygous

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written by dadhokane • dadhokane

Hello

I agree with your assumptions, but I have sequenced the CDS and would not see any difference within the CDS in both the lines. Some other reason like downstream regulatory elements or sRNAs suppressing the gene expression? Your suggestions?

Thanking you

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written by dadhokane • dadhokane

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