Examining the role of the U7 snRNA in histone pre-mRNA processing and the U7 snRNP dependent and independent roles of Lsm10 and Lsm11, two novel Lsm proteins in Drosophila Public Deposited

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  • March 21, 2019
  • Godfrey, Ashley C.
    • Affiliation: College of Arts and Sciences, Department of Biology
  • Cell cycle regulated histone gene expression ensures that the correct amounts of histones are synthesized each S phase, and is controlled in large part by the unique 3’ end of histone mRNA, which terminates in a conserved stem-loop structure generated by an endonucleolytic cleavage, rather than a polyA tail. Histone 3’ end formation involves a pre-mRNA processing reaction requiring a protein that binds the stem loop (SLBP), and the U7 snRNP, which interacts with a purine rich sequence, called the HDE (Histone Downstream Element), downstream of the cleavage site in histone pre-mRNA. The U7 snRNP is related to the spliceosomal snRNPs, small noncoding RNAs bound by a seven member Sm protein ring, but its protein ring contains two unique proteins, Lsm10 and Lsm11, and it’s only known function is in histone pre-mRNA processing. Much of this molecular model has been obtained from in vitro studies. In this thesis we characterize the U7 snRNP and its two unique proteins, Lsm10 and Lsm11, genetically and molecularly in order to determine their role in cell cycle regulated histone expression in vivo, and during development in Drosophila melanagaster. We have created null alleles of the U7 snRNA and found that they result in the production of polyadenylated histone mRNA from the use of cryptic polyadenlyation signals downstream of the normal processing site. A similar molecular phenotype also results from iii mutation of Slbp, but U7 null mutants survive to adulthood and are male and female sterile while Slbp null mutants are lethal. This difference in terminal phenotype may reflect a later onset of the histone pre-mRNA processing defect in U7 null mutants compared to Slbp null mutants. In Slbp null mutants, misprocessed histone mRNA is seen as early as the embryo stage of development while in U7 null mutants the misprocessed histone mRNA does not appear until the second instar stage of development, due to the maternal stores of U7 snRNA. We have also analyzed mutations of the Lsm10 and Lsm11 genes and found that those mutations result in the same misprocessed histone mRNA phenotype as U7 null mutants, but both mutations are lethal. We have shown that the difference in terminal phenotype is not due to an earlier onset of misprocessed histone mRNA, but instead could be due to a role(s) for Lsm10 and Lsm11 outside of histone pre-mRNA processing that is U7 independent. We have also shown that there is U7 snRNA still present in an Lsm11 null mutant. The RNA can be pulled down using TMG coupled beads suggesting that the U7 snRNA is bound by snRNP proteins even though Lsm10 and Lsm11 are not present. However this snRNA does not localize to the Histone Locus Body (HLB) suggesting that both Lsm10 and Lsm11 are required for U7 snRNP localization to the HLB.
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  • Duronio, Robert
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