In situ transcription and splicing in the Balbiani ring 3 gene.

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

Processing of pre-mRNA in polytene nuclei of Chironomus tentans salivary gland cells.

Salivary gland polytene cells in the dipteran Chironomus tentans provide exceptional experimental possibilities to analyze processing of specific pre-mRNAs in intact eukaryotic cell nuclei. Here we give a brief account of how these experimental advantages can be exploited to analyze the splicing process in vivo. In multi-intron pre-mRNAs, spliceosomes assemble and splicing is initiated cotranscriptionally for all introns. Intron excision may, however, occur mainly co- transcriptionally or mainly posttranscriptionally depending on the position of each intron in relation to the remaining transcription time and intron-specific efficiencies of excision. As measured for the U2 snRNP and an SR protein, 10-15% of the spliceosomal components are bound to pre-mRNA at active gene loci at a given moment, while the majority of the spliceosomal components are present in the nucleoplasm. A continuous redistribution of the spliceosomal components takes place in the nucleus as a result of a close coupling between transcription and spliceosomal assembly.

The intranuclear site of excision of each intron in Balbiani ring 3 pre-mRNA is influenced by the time remaining to transcription termination and different excision efficiencies for the various introns.

The 10.9-kb Balbiani ring 3 (BR3) gene contains 38 constitutively excised introns. Both nascent and nucleoplasmic, released BR3 gene pre-mRNA can be isolated by microdissection of the polytene salivary gland nuclei in which the gene is transcribed. Here we analyze the order of intron excision in relation to transcription and to intranuclear transport. We demonstrate that the introns are excised with an overall 5' to 3' polarity that is established during transcription and maintained during transport. In contrast, we also show that individual introns are excised at very different rates and that neighboring introns are removed in a preferred order that is not necessarily 5' to 3'. Splicing factors are, in addition, shown to associate with the nascent BR3 pre-mRNA. Our data argue that functional spliceosomes assemble rapidly as introns appear in the pre-mRNA, but that intron-specific properties influence the kinetics of spliceosome assembly and/or function, resulting in cotranscriptional excision of some introns, preferentially those located in the 5' part of the pre-mRNA, and posttranscriptional excision of other introns, preferentially those located in the 3' part of the pre-mRNA.