Large nuclear RNP particles--the nuclear pre-mRNA processing machine.

Processing of nuclear pre-mRNA is an important step in the regulation of gene expression and involves 5(')- and 3(')-end processing, splicing, and editing. Mammalian nuclear pre-mRNAs are assembled in large ribonucleoprotein (lnRNP) complexes, in which the entire population of nuclear pre-mRNA is individually packaged until it is exported to the cytoplasm. The lnRNP particles are supraspliceosomal complexes. They are composed of four spliceosomal substructures and an additional one, which are interconnected by the pre-mRNA, and have an overall mass of 21MDa. The additional substructure was proposed to harbor additional processing activities, such as editing components that were shown to be associated with the lnRNP particles. Here we show that the cap-binding proteins (CBPs), CBP20 and CBP80, are associated with the lnRNP particles, as well as components of the 3(')-end-processing activity. These results, together with our previous demonstration of the association of splicing factors and A-to-I editing enzymes with lnRNP particles, support the view that the lnRNP particles are the nuclear pre-mRNA processing machine. Such a machine is required to execute the nuclear processing steps of the pre-mRNA in an accurate and regulated manner. The supraspliceosomal pre-mRNA processing machine, in which each substructure represents a functional spliceosome, provides a frame onto which the pre-mRNA is folded. It allows juxtaposition of exons about to be spliced, while introns are looped out of each of the respective spliceosomes. This model can account for regulated alternative splicing, which is a major source of protein versatility in mammals.

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine.

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.