Title

Studies on intronless mRNA nucleocytoplasmic export elements

Date of Completion

January 2005

Keywords

Biology, Molecular|Biology, Cell

Degree

Ph.D.

Abstract

Splicing is coupled to and essential for the efficient nucleocytoplasmic transport of messages from intron-containing genes; however, naturally intronless messages are efficiently transported to the cytoplasm without being spliced. We are using naturally intronless genes to study the mechanism governing nucleocytoplasmic transport of unspliced messages. Previous work from the Carmichael laboratory reported that the mouse histone H2a gene contains a small element that can enhance the cytoplasmic accumulation of unspliced RNAs (Huang and Carmichael, 1997). Here we report an element within the mouse histone H3.2 coding region likewise enhances nucleocytoplasmic export of intronless beta-globin cDNA transcripts. The most active sequence is 132 nucleotides in length. This element both enhances polyadenylation, as well as cytoplasmic accumulation, of intronless beta-globin cDNA transcripts. The sequence has been further narrowed to a 22 nt element that has the same positive effects on nucleocytoplasmic export and polyadenylation as the 132 nt element. Experimental evidence suggests that binding of SR proteins correlates with export activity. These and other results support the hypothesis that SR proteins are important components of the nucleocytoplasmic mRNA transport machinery (Huang and Steitz, 2001). ^ Additional work studying the other naturally intronless genes including somatostatin transmembrane receptor 5 (SSTR5) were utilized to look at the general mechanism governing nucleocytoplasmic transport of mRNA. Here we report the identification of two RNA export elements, of ∼270 nt from SSTR5, that enhance polyadenylation and nucleocytoplasmic export of intronless beta-globin cDNA transcripts. Experimental data suggest that SR proteins most likely play a role in the activity of the second SSTR5 element, while the first element's binding partners remain unclear. Similar elements could not be identified in α-interferon and Hsp70. ^ Recently it has been reported that the phosphorylation state of SR proteins may affect export of associated mRNA (Huang et al., 2004). We report here the identification of a small nuclear protein that could potentially be a phosphatase inhibitor. The protein was first noted in a co-immunoprecipitation with αSR antibody. Mass spectrometry identified one peptide, DAVEDLESVGK, that appears in two different overlapping genes, dermcidin, and the predicted protein, protein phosphatase inhibitor-1. ^