RECURSIVE SPLICING RESOURCE PAGE

Lopez Lab - Department of Biological Sciences - Carnegie Mellon University

Background

Very large introns (>5kb) are found in many genes with important roles in development and disease (e.g. dystrophin, CFTR, Rb). Many such introns in Drosophila are processed by a recursive splicing mechanism that allows sequential removal of smaller subfragments as the intron is transcribed (Hatton et al, 1998; Burnette et al, 2005) (Figure 1). Sequence analysis and preliminary experimental data suggest that this is also the case in mammals. Conservation of the sequences and relative positions of recursive splice sites in orthologous introns among insects and among mammals suggests that recursive splicing plays an important role in expression of the corresponding genes.

Recursive splicing is mediated by special elements (recursive splice sites or "ratchetting points") that consist of juxtaposed 3' and 5' splice site sequences, with no exon between them (Figure 2). When an upstream exon is spliced to such a site, a functional 5' splice site is regenerated at the junction and can be used to splice the next segment of intron. Several ratchetting points can subdivide an intron into multiple subfragments. Because a ratchetting point is essentially a 0-nucleotide exon, recursive splicing leaves no trace in the final mRNA and can only be detected by analysis of processing intermediates or the phenotypes of mutations in ratchetting points (Burnette et al, 2005; Conklin et al, 2005).

Ratchetting points are also found at the 5' or 3' ends of some cassette exons and alternative terminal exons (Hatton et al., 1998; Burnette et al, 2005; Conklin et al, 2005). In these cases, use of the regenerated 5' splice site or initial use of the ratchetting point as a 3' splice site can be regulated developmentally so that the exon is retained in some mRNAs and removed in others (e.g., Figure 2, Figure 3).

Resources and Databases

The program Matrix Search was written to predict recursive splice sites ("RPs") using species-specific nucleotide frequency matrix models based on juxtaposition of standard 3' and 5' splice sites (Burnette et al, 2005). The matrices used can be seen here. Use the links below to access previous predictions in Drosophila and Mammals or to perform new searches on user-specified sequences. For experimentally verified sites, see: Hatton et al., 1998; Burnette et al, 2005; Conklin et al, 2005. Information on these and additional verified sites will eventually be incorporated into the databases and updated.

Code for performing searches can be obtained by sending an e-mail request to Javier Lopez.

Contact Jim Burnette or Javier Lopez for further information.

References

Burnette JM, Miyamoto-Sato E, Schaub MA, Conklin J, Lopez AJ. (2005). Subdivision of large introns in Drosophila by recursive splicing at nonexonic elements. Genetics 170(2):661-74. Supplemental Table

Conklin JF, Goldman A, Lopez AJ. (2005). Stabilization and analysis of intron lariats in vivo. Methods. 37(4):368-75.

Hatton AR, Subramaniam V, Lopez AJ. (1998). Generation of alternative Ultrabithorax isoforms and stepwise removal of a large intron by regulated resplicing at exon-exon junctions. Molecular Cell 2(6):787-96.