Pre-mRNA processing in the polytene nuclei of chironomus tentans

Abstract

Gene expression of protein encoding genes in eukaryotic cells requires extensive intranuclear maturation processing of the transcribed pre-mRNA. Three such processes are known to occur. The 5' end is capped and the 3' end is determined by cleavage and polyadenylation. A majority of pre-mRNA transcripts also contain intervening sequences, introns, that have to be removed before transport to the cytoplasm. All three processing events need protein or protein/RNA complexes. Biochemical assays in vertebrate systems and genetic analysis in yeast have identified a majority of the. factors involved in the processes and the biochemical pathway of each reaction has also been studied in some detail. Much less is known about the different processes in the intact cell nucleus, and their relationship to each other and transcription.

The aim of this thesis was to study the cell biology of the above mentioned maturation processing events in terms of functional organization of the different processing machineries in relation to each other, and to the organization of the transcription machinery in the intact nucleus of polytene Chironomus tentans cells. Polytene nuclei were studied as they provide exceptional experimental possibilities compared to diploid cells We showed that splicing factors and functional splicesomes are associated with the growing pre-mRNA transcript, and that splicing could occur on nascent transcripts. One major factor determining if an intron will be removed co- or posttranscriptionally is its location in the pre-mRNA molecule, 5' located introns are removed cotranscriptionally more frequent than 3' located ones. We also demonstrated that there is a linear polarity between the location of an intron and its probability to be removed. This is not true for closely spaced introns where factors like intron size and splice site sequences play a more predominant role. By using antibodies detecting different splicing components we demonstrated a dynamic organization of splicing factors in the cell nucleus. The majority of the different factors are located in the nucleoplasm, 80-90%, while the remaining part is associated with nascent pre-mRNA molecules.

There is a dramatic reorganization of splicing factors upon transcriptional inhibition. Splicing factors that were bound to nascent pre-mRNA leave the chromosomes and are concentrated in the nucleoplasm. The opposite was recorded when activation of specific genes were performed. Termination of BRl gene transcription occur in a region 600 bases downstream the polyadenylation site, and termination is in close relation to cleavage and polyadenylation. A small population of polyadenylated molecules could be found in the BR1 locus. These molecules have removed the final intron to a high degree (80%) compared to the nascent molecules (5-10%). In addition to this their poly(A) tail is short, only 20 adenosine residues compared to 100 residues in the nucleoplasm, indicating a slow initial phase of the addition of adenosines.