The role of exon sequences in C complex spliceosome structure.

Pre-mRNA splicing is catalyzed by a large ribonucleoprotein complex called the spliceosome. Previous electron microscopy reconstruction of C complex spliceosomes arrested between the two chemical steps of splicing revealed an averaged core structure consisting of three primary domains surrounding a central cavity. Here we characterize the involvement of pre-mRNA in this structured core of C complex by protection mapping. We find that the 3' end of the cleaved 5' exon and intron sequences flanking the branched lariat are buried in the complex. Upstream regions of the 5' exon and the entire 3' exon, including the mutant 3' splice site, are accessible and can be removed by nucleolytic cleavage. Furthermore, we show that the second-step active site of the complex, which is arrested by a 3' splice site mutation, can accept a normal 3' splice site in trans to catalyze exon ligation. Removal of the accessible exon regions alters the protein composition of the complex, but the core spliceosome proteins associated with the uridine-rich small nuclear ribonucleoproteins U2, U5, and U6 and the Prp19 complex as well as several other proteins remain intact. Two-dimensional averaged images of an exon-trimmed complex closely resemble C complex assembled on full-length pre-mRNA, supporting the hypothesis that the electron microscopy model of C complex reflects the core structure of a catalytically competent particle. Trimming the 3' exon does, however, alter the distribution of particles that appear to be missing some density, suggesting that the exon plays a role in stabilizing C complex.

The perichromosomal layer.

In addition to genetic information, mitotic chromosomes transmit essential components for nuclear assembly and function in a new cell cycle. A specialized chromosome domain, called the perichromosomal layer, perichromosomal sheath, chromosomal coat, or chromosome surface domain, contains proteins required for a variety of cellular processes, including the synthesis of messenger RNA, assembly of ribosomes, repair of DNA double-strand breaks, telomere maintenance, and apoptosis regulation. The layer also contains many proteins of unknown function and is a major target in autoimmune disease. Perichromosomal proteins are found along the entire length of chromosomes, excluding centromeres, where sister chromatids are paired and spindle microtubules attach. Targeting of proteins to the perichromosomal layer occurs primarily during prophase, and they generally remain associated until telophase. During interphase, perichromosomal proteins localize to nucleoli, the nuclear envelope, nucleoplasm, heterochromatin, centromeres, telomeres, and/or the cytoplasm. It has been suggested that the perichromosomal layer may contribute to chromosome structure, as several of the associated proteins have functions in chromatin remodeling during interphase. We review the identified proteins associated with this chromosome domain and briefly discuss their known functions during interphase and mitosis.