I did a quick search (here and elsewhere) and couldn't find anything on this subject. If all introns in a given primary transcript were spliced out in the same way, then this wouldn't matter. But lots of eukaryotic transcripts have alternate splicing patterns so that one version of the mRNA will contain coding regions that are absent in other alternate forms.

The problem relates to maintaining an open reading frame, avoiding a frame shift and the attendant (and unpredictable) consequences. If a splice donor site occurs within the 3-base codon, it might require that the two (or more) alternate acceptor sites would be in the same relative position in a related codon.

The simplest solution to this problem in my view is to make sure that all splice donor and acceptor sites would be at a codon/word boundary.

Is there any evidence that this is true?


As pointed out by others this is not true. I just verified this for humans (annotations from gencode21).


  • Obtained the start points of the CDS for all genes
  • For each exon of each gene, calculated the distance between the CDS start and exon end
  • Obtained the remainder after dividing this value by 3 (modulo)


    awk 'BEGIN{FS="\t|;";OFS="\t"} $3=="CDS"{print $4,$9}' gencode.v21.annotation.gtf > cdsstart.txt 

    awk 'BEGIN{FS="\t|;";OFS="\t"} NR==FNR{a[$2]=$1;next} ($3=="exon" && $9 in a){if($4>a[$9]) q[($5-a[$9])%3]++} END{for(i in q) print i,q[i]}' cdsstart.txt gencode.v21.annotation.gtf


position | # cases
   -2    | 101191
   -1    | 100954
    0    | 99499

As you can see the exons do not necessarily terminate at the third position of the codon.


Here is exon size data of some random gene (human ROR-gamma). Link: http://www.ncbi.nlm.nih.gov/nuccore/NM_005060.3

It gets cut at all three possible positions of codon:

exon 2 30

exon 3 86

exon 4 142

exon 5 513

exon 6 122

exon 7 133

exon 8 108

exon 9 111

exon 10 110

Alernatively spliced isoform B of this gene gets different first exon. At the junction codon ACA (isoform A) gets replaced with codon TCA (isoform B).


I don't have time to find examples right now but no, it's not true. You often get cases like this (lower case letters represent the intron):


Which would be spliced to:


Note that the second codon consists of one nucleotide from exon1 and two from exon2. I'll try and update this with real world examples, but I can assure you it is possible and I have seen it quite often.


As Alex M shows, many lengths of exons are 3n+1 or +2. It seems cells do not care about in-frame.

In some cases, exon insertion by alternative splicing shift the frame and produce a short version of gene product or introduce premature stop codon (PTC). When PCT is recognized in cells, the spliced transcript is quickly degraded. This is called nonsense mediated decay (NMD).

Splicing machinery does not care about frame of CDS, and this makes various gene regulations possible.


This review shows an example of NMD by alternative splicing. See the description about caspase-2.


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