Recursive Splicing: Carnegie Mellon University Research Reveals How Cells Process Large Genes

"While some scientists have suspected that large introns mightnot be removed in one piece through direct splicing, no one hadidentified how this could happen. Now we have identified a way," saidAntonio-Javier Lopez, professor of biological sciences at CarnegieMellon. Ultimately, recursive splicing could be responsible forthwarting molecular mishaps in the expression of large human genesassociated with diseases like muscular dystrophy, cystic fibrosis andcancer.

"We found that many large introns are removed by multiplerecursive splicing steps," Lopez said. "These steps involve thesequential excision of smaller subfragments. Our work also indicatesthat most recursive splicing events leave no clues in the final mRNA.This is why they have not been detected before now."

These previously undetected events could have profoundimplications for predicting what constitutes a gene and for studyinggene expression, mutation and evolution, according to Lopez.

For example, recursive splicing must now be taken into accountwhen evaluating mutations that disrupt gene expression and produce adysfunctional or non-functional protein.

"Current data indicate that at least 15 percent ofdisease-causing mutations occur at standard signals where intronremoval takes place through direct splicing. Mutations at recursivesplice sites may cause additional diseases, but until now we haven'tlooked for them."

Knowledge of recursive splicing also will help investigatorspredict structures of genes that span large intervals of DNA, Lopezsaid.

Recursive splicing relies on the unusual activity of aratchetting point, a pattern of chemical groups (nucleotides)previously discovered within the genome by Lopez. One end of aratchetting point contains a sequence of nucleotides similar to thesignal normally found at the beginning of an intron. This signal isjuxtaposed with another sequence like that normally found at the end ofan intron. Such a unique pairing allows a ratchetting point to functionsequentially as an acceptor for splicing to an upstream exon and thenas a donor for splicing to the next downstream ratchetting point orexon. As the process goes from ratchetting point to ratchetting point,small signature loops of RNA called lariats are released from theintron. Repeated over and over, recursive splicing eventually binds, orligates, two distant exons.

Lopez's team developed molecular tools to analyze the lariatsreleased from any intron during splicing in vivo. In his analyses, hefound that the production of recursive lariats greatly exceeded that ofdirect lariats, indicating that recursive splicing is the predominantprocessing pathway for long introns. Lopez combined these experimentaldata with computational and phylogenetic analyses of several fruitflyand other insect species.

"Our experimental results agreed with the computationalfindings, indicating that these ratchetting points mediate the removalof intron subfragments in one direction as the gene is transcribedinitially from DNA into RNA," Lopez said.

Lopez found that predicted recursive splice sites were 10times more likely than expected to be found in introns greater than 200kilobases in length, and 92 percent of them were conserved over atleast 25 million years of insect evolution. This discovery stronglysuggests that recursive splicing plays a special role in the correctexpression of large genes. Bioinformatic and phylogenetic analysesconducted by Lopez also indicate that recursive splicing plays a rolein at least 124 fruitfly introns, with up to seven potential cuttingsteps identified for a single intron. Similar analyses suggest that thesame process also occurs in large introns of mammals, including humans,and the Lopez team is now testing this hypothesis experimentally.

"The striking evolutionary conservation of ratchetting pointssuggests that recursive splicing provides specific advantages for largeintrons," Lopez said. "One possibility is that recursive splicingprevents the generation of long RNA transcripts that could formstructures that interfere with correct processing into mRNA. Another isthat recursive splicing might help stimulate transcription through longintrons by promoting interactions between the splicing andtranscription machineries. We also know already that recursive splicingis used to control the removal of certain exons from mRNAs, generatingstructural and functional variation among the gene products."

This research is funded by the National Institutes of Health and the Pa. Department of Health.