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J. Biol. Chem., Vol. 279, Issue 50, 52437-52446, December 10, 2004

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Genome-wide Analysis of Pre-mRNA Splicing

INTRON FEATURES GOVERN THE REQUIREMENT FOR THE SECOND-STEP FACTOR, Prp17 IN SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE^*

Aparna K. Sapra, Yoav Arava¶, Piyush Khandelia, and Usha Vijayraghavan||

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India and the Department of Biochemistry, Stanford University, Stanford, California 94305-5307

Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a 13-nucleotide spacing between their branch point nucleotide and 3 ' splice site. In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.

Received for publication, August 3, 2004

^* This work was supported in part by an International Senior Research Fellowship from The Wellcome Trust, United Kingdom (to U. V.); by partial infrastructure support from the Indian Council of Medical Research, Government of India; by scholarships (to A. K. S. and P. K.) from Council of Scientific and Industrial Research, Government of India; and by an UNESCO-ASM (2001) short term travel fellowship (to A. K. S.), which facilitated the microarray experiments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

This article was selected as a Paper of the Week.

^¶ Research associate of the Howard Hughes Medical Institute. Present address: Dept. of Biology, Technion, Haifa 32000, Israel.

^|| To whom correspondence should be addressed. Tel.: 91-80-2360-0168; Fax: 91-80-2360-2697; E-mail: uvr{at}mcbl.iisc.ernet.in or usha_vijayraghavan{at}yahoo.co.uk.

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