Poster abstracts

Poster number 83 submitted by Lilia Turcios

Co-Transcriptional cleavage in the immunoglobin M (IgM) gene

Lilia Turcios (Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky), Martha Peterson (Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky)

Abstract:
Cotranscriptional cleavage events, driven by specific cis-acting elements known as CoTC, are required downstream of some poly(A) signals to terminate transcription. Little is known about these elements, but in some cases, a pause site can replace the CoTC element. The IgM gene is alternatively processed to produce two mRNA isoforms by either splicing the primary transcript between the Cµ4 and the M1 exons (splice) or cleaving and polyadenylating it at the µs poly(A) signal (pA); the pA/splice RNA expression ratio increases during B cell maturation. A pause site was identified downstream of the µs pA signal, but it is not known whether the transcript also contains a CoTC element. To explore whether a CoTC-like element exists within the IgM gene that may contribute to developmental changes in Ig expression, we analyzed the RNA downstream of the µspA signal by semi-quantitative RT-PCR using sets of primers that span 4 Kb of sequence downstream of the µspA signal. The presence of a CoTC element would be detected as an abrupt decrease in transcript somewhere downstream of the ìspA signal. However, we observed a gradual decrease in transcripts over the region in both B cells and plasma cells, suggesting a natural CoTC element is not present. To examine the effect a CoTC element would have on the competition between the splice and cleavage-polyadenylation reactions, we inserted the B-globin CoTC sequence into two locations downstream of the µspA signal, both in the presence and absence of the pause site. We observed that inclusion of the B-globin CoTC element toward the end of the intron causes cotranscriptional cleavage but does not greatly affect the RNA processing reactions, except as expected due to the size of the insert. However, inserting the B-globin CoTC element close to the µspA site not only caused cleavage and early termination of transcription, but also caused an increase in the pA/splice ratio. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts.

Keywords: Co-transcriptional Cleavage, IgM, RNA processing