We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif-finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within ±2 residues from the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http://www.cbs.dtu.dk/services/ChloroP/.

Histone pre-mRNA 3′ processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA′CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3′ processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5′ of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3′ position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614–2620].