Is exon order always preserved in splicing?

Question

Are there any cases in which the splicing machinery constructs an mRNA in which the exons are not in the 5' -> 3' genomic order? I'm interested any such cases, whether they involve constitutive or alternative splicing.

Answer 1

I don't have any literature to back this up but I doubt that it occurs (at least frequently).

For example, imagine a simple three exon gene. Upon splicing exon 1 to exon 3, exon 2 would be excised as part of the intron lariat and subsequently degraded. So in order for exon 2 to be spliced to exon three you would need to either have splicing between exon 3 and exon 2 in the lariat or another copy of the pre-mRNA. This is typically called trans-splicing but it only occurs in specialized systems such as spliced-leader sequences in C. elegans.

Answer 2

There are references in the literature to the phenomenon of "exon scrambling" which seems to be what you are asking about, but the prevailing view is that the evidence for this process, which comes from comparing EST sequences with genome sequences, can be explained by cloning artefacts occurring during EST characterisation. Certainly I agree that there is no evidence for a gene in which the rearrangement of exons is part of the normal pathway for mRNA generation.

Shao et al. (2006) Bioinformatic analysis of exon repetition, exon scrambling and trans-splicing in humans. Bioinformatics 22: 692-698

There is also more recent evidence from deep sequencing for rearrangement of exon order via circular RNAs:

Salzman et al. (2012), Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLOS ONE 7: e30733   DOI: 10.1371/journal.pone.0030733   

Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells.

Incidentally it is perhaps worth mentioning that in the case of the lectin concanavalin A the equivalent effect is produced post-translationally when the protein rearranges.

Carrington et al. (1985) Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature 313:64-67

Answer 3

After performing a quick literature search, I am, as with GWW, unable to provide any literature against this occurring, although this paper by Black (2005) states that exons in multi-exon pre-mRNAs are always maintained in order.

Black DL. 2005. A simple answer for a splicing conundrum. Proceedings of the National Academy of Sciences of the United States of America 102: 4927–8..

Separate components of the spliceosome recognise sequences at the the start and end of each intron, along with a branch point adenine and some other conserved (highly in the case of yeast) regions upstream of the 3' end. The components seem to assemble in a particular order, which is 5' to 3' directional.

The general consensus seems to be that the spliceosome moves along the pre-mRNA from 5' to 3' removing the introns and splicing the exons together to form the mature transcript, ready for translation. The introns are subsequently degraded. In the case of eukaryotes some processing is required to transport the mRNA out of the nucleus to the ribosomes in the cytoplasm.

The mRNA would have a start codon near the 5' terminus and stop codon near the 3' end, at the boundaries of the CDS, and it isn't clear to me how this structure could be maintained if exon order didn't follow the 5' to 3' assemblage. I should imagine that the protein could be affected in much a similar way to exon skipping, possibly resulting in a truncated, inactive or ineffective protein. As protein folding is determined by the interactions between the amino acids, any change in exon order (secondary structure) is likely to result in a different tertiary structure.