Gerstner Sloan Kettering (GSK) student Brian Joseph has been awarded the Chairman’s Prize for his paper “Short cryptic exons mediate recursive splicing in drosophila,” published in Nature Structural and Molecular Biology in April 2018. His work sheds light on the mechanism and landscape of recursive splicing (RS).
The $2,000 Chairman’s Prize, which is awarded annually, was established by GSK Board of Trustees Chair Louis V. Gerstner, Jr., for whom the school is named. Mr. Gerstner is an internationally renowned corporate leader who has long been an advocate of quality education.
Here, Mr. Joseph, a sixth-year student in the laboratory of developmental biologist Eric Lai, describes his winning paper.
In most eukaryotic genes, protein-coding exons are interrupted by one or more noncoding introns. For such genes, protein production requires the accurate removal of introns from messenger RNA (mRNA) by splicing. Therefore, the choice of splice site (SS) and the efficiency of intron splicing can be regulated to influence gene output.
SS selection may be obvious in the context of small introns that measure less than 200 nucleotides (nt). However, it gets incredibly complicated within long introns that measure more than 10,000 nt and contain many cryptic sites.
My colleagues and I are particularly fascinated by long introns because they are found in developmental genes and generate transcriptional diversity through alternative splicing. However, they are technically challenging to study in their natural context, and fundamental questions regarding their processing remain unanswered.
My work focuses on recursive splicing, a mechanism through which a large intron is removed in two or more smaller segments. This process requires special splice sites called ratchet points (RPs), which consist of tandem splice acceptor and donor sequences and resemble 0-nt exons.
Taking a Closer Look at Recursive Splicing
We used several approaches to investigate how recursive splicing works in the fruit fly Drosophila melanogaster.
First, we used CRISPR/Cas9 technology to disrupt RP splice donors in endogenous genes in the fly and found that all mutant alleles produced mRNA containing novel, unannotated, cryptic exons. The addition of these cryptic exons was catastrophic and, in some cases, even lethal to the animal. We leveraged these observations to examine the mechanism of recursive splicing. Indeed, our cell culture tests revealed that these unannotated, cryptic exons are essential for recursive splicing.
We then used computational methods to mine nascent RNA sequencing data sets and vastly increased the known annotations of RPs. With these data, we found that the majority of RPs are contained within cryptic exons, extending our experimental findings across the fly’s entire genome. We also expanded the set of known expressed cassette exons that likely undergo recursive splicing. Last, we uncovered an exon length that is unexpectedly common among all exons flanked by long introns, whether recursively processed or not.
This paper is the first to describe RS site disruptions, which has resulted in the reclassification of 0-nt exon RPs as larger cryptic RP-exons. These exons are constitutively spliced into but are never included in the final transcript, revealing that RS is a form of alternative splicing. Overall, our work highlights the potential of RS as a checkpoint in gene regulation.
Our research provides three ideas that form the basis for future work on long introns and RS biology:
- Long introns correlate with a shorter exon size, regardless of recursive or canonical splicing. Further tests are required to examine whether this is a coincidence or a real constraint set by intron architecture.
- RPs subdivide long introns linearly, suggesting that there is an optimal intron length for efficient splicing. We are currently testing this hypothesis by examining splicing kinetics.
- Finally, we are interested in testing the consequences of deleting RPs on long intron processing and endogenous gene function.
We plan to explore these ideas using a combination of genetics, molecular biology, and bioinformatics. Overall, these studies will help us better understand how gene architecture influences gene expression.