Originally published as Genetics Published Articles Ahead of Print on March 31, 2005.

Genetics, Vol. 170, 661-674, June 2005, Copyright © 2005
doi:10.1534/genetics.104.039701

Subdivision of Large Introns in Drosophila by Recursive Splicing at Nonexonic Elements

James M. Burnette^*, Etsuko Miyamoto-Sato, Marc A. Schaub^*,, Jamie Conklin^* and A. Javier Lopez^*,1

^* Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan
School of Computer Science, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

¹ Corresponding author: Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213.
E-mail: jlaa{at}andrew.cmu.edu

Many genes with important roles in development and disease contain exceptionally long introns, but special mechanisms for their expression have not been investigated. We present bioinformatic, phylogenetic, and experimental evidence in Drosophila for a mechanism that subdivides many large introns by recursive splicing at nonexonic elements and alternative exons. Recursive splice sites predicted with highly stringent criteria are found at much higher frequency than expected in the sense strands of introns >20 kb, but they are found only at the expected frequency on the antisense strands, and they are underrepresented within introns <10 kb. The predicted sites in long introns are highly conserved between Drosophila melanogaster and Drosophila pseudoobscura, despite extensive divergence of other sequences within the same introns. These patterns of enrichment and conservation indicate that recursive splice sites are advantageous in the context of long introns. Experimental analyses of in vivo processing intermediates and lariat products from four large introns in the unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these introns are removed by a series of recursive splicing steps using the predicted nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also confirmed that recursive splicing is the predominant processing pathway even with a shortened version of the intron. We discuss currently known and potential roles for recursive splicing.

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