3-DIMENSIONAL IMAGE-RECONSTRUCTION OF LARGE NUCLEAR RNP (LNRNP) PARTICLES BY AUTOMATED ELECTRON TOMOGRAPHY

Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are pack aged in multi-component large nuclear ribonucleoprotein (lnRNP) partic les. These lnRNP particles, which sediment at the 200 S region in sucr ose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factor s, including U2AF and the SR proteins. Electron microscopy of lnRNP pa rticles revealed a large compact structure of 50 nm in diameter. In th is study we employed automated computed tomography from electron micro graphs for the three-dimensional (3D) image reconstruction of individu al lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed acc ording to the back projection principle. For rendering, real time disp lay and comparison of the reconstructed particles, interactive compute r graphics was employed. The reconstructed 3D images show a compact st ructure composed of four major subunits connected to each other. Compa rison of the reconstructed lnRNP particles revealed morphological simi larity of the individual particles, as well as similarity among the su b-structures. Based on these observations we propose a model for the p ackaging of nuclear pre-mRNAs in lnRNP particles where each sub-struct ure represents a functional unit. This model is compatible with the re quirements for alternative splicing in multi-intronic pre-mRNAs, and w ith the fact that the splicing of multi-intronic pre-mRNAs does not oc cur in a sequential manner. (C) 1997 Academic Press Limited.