A REGION OF DROSOPHILA SLBP DISTINCT FROM THE HISTONE PRE-MRNA BINDING AND PROCESSING DOMAINS IS ESSENTIAL FOR DEPOSITION OF HISTONE MRNA IN THE OOCYTE

Metazoan histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. Instead they end in a 3’end Stemloop (SL). Processing of the histones pre-mRNAs is accomplished by an endonucleolytic cleavage after the SL. The stem loop binding protein (SLBP) binds to the SL, and SLBP is a key factor in all steps of the life cycle of histone mRNAs. We are studying the role of SLBP in Drosophila melanogaster in vivo. In Drosophila each histone gene contains a cryptic polyA site after the histone processing site, and when histone pre-mRNA processing is defective histone mRNAs are polyadenylated. Using FLY-CRISPRCas9 we obtained a 11 deletion (SLBP∆11) null mutant and a 30 nucleotide deletion (SLBP∆30) in the N-terminal domain (NTD) of SLBP. The 30nt deletion removed 10aa from the N-terminal domain of SLBP in a region of unknown function distinct from the processing domain. The SLBP∆30 mutant was viable, but females were sterile. They laid eggs, but the eggs didn’t hatch, because they didn’t store histone mRNA in the egg. In Drosophila nurse cells produce large amounts of histone mRNA at the end of oogenesis which is translated and stored in the egg to allow the development of the embryo until zygotic histone gene transcription turns on. The stored histone mRNA is produced in absence of DNA replication. Despite having normal amount of SLBP, the SLBP∆30 mutant expresses small amounts of polyadenylated histone mRNA at all stages. In the ovary histone mRNA expression is normal in the rapidly replicating nurse cells throughout oocyte development but very little histone mRNA is expressed at the end of oogenesis after nurse cell replication is completed. Immunofluorescence data shows that the SLBP∆30 protein is mainly localized in the cytoplasm at this stage, suggesting the deleted region is important for nuclear import of SLBP at the end of oogenesis. The histone locus bodies present at the histone genes are also defective, and do not activate transcription of the histone genes. These results suggest that defective nuclear import of SLBP∆30 may lead to a defect in HLBs and histone gene transcription.Doctor of Philosoph