Biochemical and structural studies of pre-mRNA splicing

Abstract

When eucaryotic genes are expressed, they are transcribed into pre-mRNAs (precursor mRNAs) that need extensive processing before they can be exported from the nucleus as mRNAs and translated into proteins in the cytoplasm. One central processing event is splicing, which means removal of intervening, non-coding sequences, introns and joining of functional sequences, exons. Splicing involves more than 50 different proteins and five snRNAs (small nuclear RNAs) that together form the active splicing complex, the spliceosome.

This thesis focuses on how splicing is temporally and spatially coupled to transcription inside the intact cell nucleus. The model gene is BR3 (Balbiani Ring 3) which has 38 introns; and is highly expressed in the salivary glands of the dipteran Chironomus tentans. Using microdissection of the nascent BR3 transcripts and RT-PCR (reverse transcription coupled to polymerase chain reaction), we have shown that splicing is closely coupled to transcription in vivo. Once an intron has been transcribed, the splicing machinery recognizes it and starts to work. There is a general 5' to 3' polarity of splicing; i.e., introns that are transcribed early are co-transcriptionally excised to a higher extent than introns that are transcribed late. However, a given intron is influenced by various local properties, meaning that neighboring introns can be removed in a non-random, non-5' to 3' order.

We have further reconstructed the 3D structure of the active BR3 gene with electron tomography. Using antibodies against RNA polymerase II and a splicing factor, U2 snRNP (U2 small nuclear ribonucleoprotein), we found that both are present in the same, dynamic complex. We call these complexes NTS complexes for nascent transcript and splicing complex. As the NTS complex moves along the gene there are extensive changes in overall shape, substructure and molecular mass (4.6-7.3 MDa) within it. Maximally one complete spliceosome is assembled on the multi-intron transcript at a given timepoint and the spliceosome is not a structurally well-defined unit in situ but rather a functional entity. During transcript elongation, spliceosomal. factors are probably continuously added to and released from the NTS complex. A full cycle of spliceosome formation and catalysis takes place in less than 25 seconds.

Finally, we have cloned and characterized a novel SR-like protein, Ct-RSF (Chironomus tentans Repressing Splicing Factor). SR (Serine-Arginine rich) proteins are splicing factors important for both constitutive and alternative splicing. In contrast to SR proteins, Ct-RSF represses in vitro splicing. Ct-RSF represses an early step of spliceosomal formation. In vivo, CtRSF binds extensively to BR1 and BR2 (Balbiani Ring 1 and 2 respectively) pre-mRNAs, which mainly consist of exon sequences, and little to BR3 pre-mRNA, half of which are introns. In contrast, the splicing factors U1 and U2 snRNPs bind more to BR3 than to BR1 and BR2 premRNAs. Our hypothesis is that Ct-RSF binds to exons sequences and represses splicing at cryptic splice sites.