Differences and similarities between Drosophila and mammalian 3′ end processing of histone pre-mRNAs

Abstract

We used nuclear extracts from Drosophila Kc cells to characterize 3′ end processing of Drosophila histone pre-mRNAs. Drosophila SLBP plays a critical role in recruiting the U7 snRNP to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem–loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem–loop and the HDE does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem–loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3′ OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2′O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5′ end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3′ end processing between vertebrates and invertebrates.