AUTOMATED ELECTRON TOMOGRAPHY OF LARGE NUCLEAR RNP (INRNP) PARTICLES - THE NATURALLY ASSEMBLED COMPLEXES OF PRECURSOR MESSENGER-RNA AND SPLICING FACTORS

Splicing of nuclear pre-mRNA is an important step in the regulation of gene expression as only correctly spliced mRNAs will be exported to t he cytoplasm to function in protein synthesis, Nuclear RNA transcripts of split genes and their splicing products, as well as the general po pulation of nuclear polyadenylated RNA, ape packaged in multicomponent large nuclear ribonucleoprotein (lnRNP) particles. These InRNP partic les, which sediment at the 200S region in sucrose gradients, contain a ll U snRNPs required for pre-mRNA splicing and several protein splicin g factors, including U2AF and the SR proteins and can thus be viewed a s naturally assembled complexes of pre-mRNA and splicing factors, We h ave previously reconstructed. the three-dimensional image of negativel y stained individual lnRNP particles by automated electron tomography. The reconstruction revealed a compact structure, 50 nm in diameter, c omposed of four major subunits, Here we further analyzed the reconstru cted models and the apparent connectivity between the subunits using a new rendering technique, The uniformity of the lnRNP particles was sub stantiated by measurement of the volume engulfed by their surface, Thi s study further supports the model proposed for the packaging of nucle ar pre-mRNAs in lnRNP particles, where each substructure represents a functional unit. This model is compatible with the requirements for al ternative splicing in multiintronic pre-mRNAs, and with the fact that the splicing of multiintronic pre-mRNAs does not occur in a sequential manner. (C) 1997 Academic Press.